DNS encoding stem cell factor

ABSTRACT

Novel stem cell factors, oligonucleotides encoding the same, and methods of production, are disclosed. Pharmaceutical compositions and methods of treating disorders involving blood cells are also disclosed.

This is a continuation of application Ser. No. 07/982,255 filed on Nov.25, 1992, now abandoned. This is a continuation-in-part application ofSer. No. 07/589,701, filed Oct. 1, 1990 now abandoned which is acontinuation-in-part application of Ser. No. 07/573,616 filed Aug. 24,1990 now abandoned which is a continuation-in-part application of Ser.No. 07/537,198 filed Jun. 11, 1990 now abandoned which is acontinuation-in-part application of Ser. No. 07/422,383 filed Oct. 16,1989 now abandoned hereby incorporated by reference.

The present invention relates in general to novel factors whichstimulate primitive progenitor cells including early hematopoieticprogenitor cells, and to DNA sequences encoding such factors. Inparticular, the invention relates to these novel factors, to fragmentsand polypeptide analogs thereof and to DNA sequences encoding the same.

BACKGROUND OF THE INVENTION

The human blood-forming (hematopoietic) system is comprised of a varietyof white blood cells (including neutrophils, macrophages, basophils,mast cells, eosinophils, T and B cells), red blood cells (erythrocytes)and clot-forming cells (megakaryocytes, platelets).

It is believed that small amounts of certain hematopoietic growthfactors account for the differentiation of a small number of “stemcells” into a variety of blood cell progenitors for the tremendousproliferation of those cells, and for the ultimate differentiation ofmature blood cells from those lines. The hematopoietic regenerativesystem functions well under normal conditions. However, when stressed bychemotherapy, radiation, or natural myelodysplastic disorders, aresulting period during which patients are seriously leukopenic, anemic,or thrombocytopenic occurs. The development and the use of hematopoieticgrowth factors accelerates bone marrow regeneration during thisdangerous phase.

In certain viral induced disorders, such as acquired autoimmunedeficiency (AIDS) blood elements such as T cells may be specificallydestroyed. Augmentation of T cell production may be therapeutic in suchcases.

Because the hematopoietic growth factors are present in extremely smallamounts, the detection and identification of these factors has reliedupon an array of assays which as yet only distinguish among thedifferent factors on the basis of stimulative effects on cultured cellsunder artificial conditions.

The application of recombinant genetic techniques has clarified theunderstanding of the biological activities of individual growth factors.For example, the amino acid and DNA sequences for human erythropoietin(EPO), which stimulates the production of erythrocytes, have beenobtained. (See, Lin, U.S. Pat. No. 4,703,008, hereby incorporated byreference). recombinant methods have also been applied to the isolationof cDNA for a human granulocyte colony-stimulating factor, G-CSF (See,Souza, U.S. Pat. No. 4,810,643, hereby incorporated by reference), andhuman granulocyte-macrophage colony stimulating factor (GM-CSF) [Lee, etal., Proc. Natl. Acad. Sci. USA, 82, 4360-4364 (1985); Wong, et al.,Science, 228, 810-814 (1985)], murine G- and GM-CSF [Yokota, et al.,Proc. Natl. Acad. Sci. (USA), 81, 1070 (1984); Fung, et al., Nature,307, 233 (1984); Gough, et al., Nature, 309, 763 (1984)], and humanmacrophage colony-stimulating factor (CSF-1) [Kawasaki, et al., Science,230, 291 (1985)].

The High Proliferative Potential Colony Forming Cell (HPP-CFC) assaysystem tests for the action of factors on early hematopoieticprogenitors [Zont, J. Exp. Med., 159, 679-690 (1984)]. A number ofreports exist in the literature for factors which are active in theHPP-CFC assay. The sources of these factors are indicated in Table 1.The most well characterized factors are discussed below.

An activity in human spleen conditioned medium has been termedsynergistic factor (SF). Several human tissues and human and mouse celllines produce an SF, referred to as SF-1, which synergizes with CSF-1 tostimulate the earliest HPP-CFC. SF-1 has been reported in mediaconditioned by human spleen cells, human placental cells, 5637 cells (abladder carcinoma cell line), and EMT-6 cells (a mouse mammary carcinomacell line). The identity of SF-1 has yet to be determined. Initialreports demonstrate overlapping activities of interleukin-1 with SF-1from cell line 5637 [Zsebo et al., Blood, 71, 962-968 (1988)]. However,additional reports have demonstrated that the combination ofinterleukin-1 (IL-1) plus CSF-1 cannot stimulate the same colonyformation as can be obtained with CSF-1 plus partially purifiedpreparations of 5637 conditioned media [McNiece, Blood, 73, 919 (1989)].

The synergistic factor present in pregnant mouse uterus extract isCSF-1. WEHI-3 cells (murine myelomonocytic leukemia cell line) produce asynergistic factor which appears to be identical to IL-3. Both CSF-1 andIL-3 stimulate hematopoietic progenitors which are more mature than thetarget of SF-1.

Another class of synergistic factor has been shown to be present inconditioned media from TC-1 cells (bone marrow-derived stromal cells).This cell line produces a factor which stimulates both early myeloid andlymphoid cell types. It has been termed hemolymphopoietic growth factor1 (HLGF-1). It has an apparent molecular weight of 120,000 [McNiece etal., Exp. Hematol., 16, 383 (1988)].

Of the known interleukins and CSFs, IL-1, IL-3, and CSF-1 have beenidentified as possessing activity in the HPP-CFC assay. The othersources of synergistic activity mentioned in Table 1 have not beenstructurally identified. Based on the polypeptide sequence andbiological activity profile, the present invention relates to a moleculewhich is distinct from IL-1, IL-3, CSF-1 and SF-1.

TABLE 1 Preparations Containing Factors Active in the HPP-CFC AssaySource¹ Reference Human Spleen CM [Kriegler, Blood, 60, 503 (1982)]Mouse Spleen CM [Bradley, Exp. Hematol. Today Baum, ed., 285 (1980)] RatSpleen CM [Bradley, supra, (1980)] Mouse lung CM [Bradley, supra,(1980)] Human Placental CM [Kriegler, supra (1982)] Pregnant MouseUterus [Bradley, supra (1980)] GTC-C CM [Bradley, supra (1980)] RH3 CM[Bradley, supra (1980)] PHA PBL [Bradley, supra (1980)] WEHI-3B CM[McNiece, Cell Biol. Int. Rep., 6, 243 (1982)] EMT-6 CM [McNiece, Exp.Hematol., 15, 854 (1987)] L-Cell CM [Kriegler, Exp. Hematol., 12, 844(1984)] 5637 CM [Stanley, Cell, 45, 667 (1986)] TC-1 CM [Song, Blood,66, 273 (1985)] ¹CM = Conditioned media.

When administered parenterally, proteins are often cleared rapidly fromthe circulation and may therefore elicit relatively short-livedpharmacological activity. Consequently, frequent injections ofrelatively large doses of bioactive proteins may be required to sustaintherapeutic efficacy. Proteins modified by the covalent attachment ofwater-soluble polymers such as polyethylene glycol, copolymers ofpolyethylene glycol and polypropylene glycol, carboxymethyl cellulose,dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline areknown to exhibit substantially longer half-lives in blood followingintravenous injection than do the corresponding unmodified proteins[Abuchowski et al., In: “Enzymes as Drugs”, Holcenberg et al., eds.Wiley-Interscience, New York, N.Y., 367-383 (1981), Newmark et al., J.Appl. Biochem. 4:185-189 (1982), and Katre et al., Proc. Natl. Acad.Sci. USA 84, 1487-1491 (1987)]. Such modifications may also increase theprotein's solubility in aqueous solution, eliminate aggregation, enhancethe physical and chemical stability of the protein, and greatly reducethe immunogenicity and antigenicity of the protein. As a result, thedesired in vivo biological activity may be achieved by theadministration of such polymer-protein adducts less frequently or inlower doses than with the unmodified protein.

Attachment of polyethylene glycol (PEG) to proteins is particularlyuseful because PEG has very low toxicity in mammals [Carpenter et al.,Toxicol. Appl. Pharmacol., 18, 35-40 (1971)]. For example, a PEG adductof adenosine deaminase was approved in the United States for use inhumans for the treatment of severe combined immunodeficiency syndrome. Asecond advantage afforded by the conjugation of PEG is that ofeffectively reducing the immunogenicity and antigenicity of heterologousproteins. For example, a PEG adduct of a human protein might be usefulfor the treatment of disease in other mammalian species without the riskof triggering a severe immune response.

Polymers such as PEG may be conveniently attached to one or morereactive amino acid residues in a protein such as the alpha-amino groupof the amino-terminal amino acid, the epsilon amino groups of lysineside chains, the sulfhydryl groups of cysteine side chains, the carboxylgroups of aspartyl and glutamyl side chains, the alpha-carboxyl group ofthe carboxyl-terminal amino acid, tyrosine side chains, or to activatedderivatives of glycosyl chains attached to certain asparagine, serine orthreonine residues.

Numerous activated forms of PEG suitable for direct reaction withproteins have been described. Useful PEG reagents for reaction withprotein amino groups include active esters of carboxylic acid orcarbonate derivatives, particularly those in which the leaving groupsare N-hydroxysuccinimide, p-nitrophenol, imidazole or1-hydroxy-2-nitrobenzene-4-sulfonate. PEG derivatives containingmaleimido or haloacetyl groups are useful reagents for the modificationof protein free sulfhydryl groups. Likewise, PEG reagents containingamino, hydrazine or hydrazide groups are useful for reaction withaldehydes generated by periodate oxidation of carbohydrate groups inproteins.

It is an object of the present invention to provide a factor causinggrowth of early hematopoietic progenitor cells.

SUMMARY OF THE INVENTION

According to the present invention, novel factors, referred to herein as“stem cell factors” (SCF) having the ability to stimulate growth ofprimitive progenitors including early hematopoietic progenitor cells areprovided. These SCFs also are able to stimulate non-hematopoietic stemcells such as neural stem cells and primordial germ stem cells. Suchfactors include purified naturally-occurring stem cell factors. Theinvention also relates to non-naturally-occurring polypeptides havingamino acid sequences sufficiently duplicative of that ofnaturally-occurring stem cell factor to allow possession of ahematopoietic biological activity of naturally occurring stem cellfactor.

The present invention also provides isolated DNA sequences for use insecuring expression in procaryotic or eukaryotic host cells ofpolypeptide products having amino acid sequences sufficientlyduplicative of that of naturally-occurring stem cell factor to allowpossession of a hematopoietic biological activity of naturally occurringstem cell factor. Such DNA sequences include:

(a) DNA sequences set out in FIGS. 14B, 14C, 15B, 15C, 15D, 42 and 44 ortheir complementary strands;

(b) DNA sequences which hybridize to the DNA sequences defined in (a) orfragments thereof; and

(c) DNA sequences which, but for the degeneracy of the genetic code,would hybridize to the DNA sequences defined in (a) and (b).

Also provided are vectors containing such DNA sequences, and host cellstransformed or transfected with such vectors. Also comprehended by theinvention are methods of producing SCF by recombinant techniques, andmethods of treating disorders. Additionally, pharmaceutical compositionsincluding SCF and antibodies specifically binding SCF are provided.

The invention also relates to a process for the efficient recovery ofstem cell factor from a material containing SCF, the process comprisingthe steps of ion exchange chromatographic separation and/or reversephase liquid chromatographic separation.

The present invention also provides a biologically-active adduct havingprolonged in vivo half-life and enhanced potency in mammals, comprisingSCF covalently conjugated to a water-soluble polymer such aspolyethylene glycol or copolymers of polyethylene glycol andpolypropylene glycol, wherein said polymer is unsubstituted orsubstituted at one end with an alkyl group. Another aspect of thisinvention resides in a process for preparing the adduct described above,comprising reacting the SCF with a water-soluble polymer having at leastone terminal reactive group and purifying the resulting adduct toproduce a product with extended circulating half-life and enhancedbiological activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an anion exchange chromatogram from the purification ofmammalian SCF.

FIG. 2 is a gel filtration chromatogram from the purification ofmammalian SCF.

FIG. 3 is a wheat germ agglutinin-agarose chromatogram from thepurification of mammalian SCF.

FIG. 4 is a cation exchange chromatogram from the purification ofmammalian SCF.

FIG. 5 is a C₄ chromatogram from the purification of mammalian SCF.

FIG. 6 shows sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis (PAGE) (SDS-PAGE) of C₄ column fractions from FIG. 5.

FIG. 7 is an analytical C₄ chromatogram of mammalian SCF.

FIG. 8 shows SDS-PAGE of C₄ column fractions from FIG. 7.

FIG. 9 shows SDS-PAGE of purified mammalian SCF and deglycosylatedmammalian SCF.

FIG. 10 is an analytical C₄ chromatogram of purified mammalian SCF.

FIG. 11 shows the amino acid sequence of mammalian SCF derived fromprotein sequencing.

FIG. 12 shows

A. oligonucleotides for rat SCF cDNA

B. oligonucleotides for human SCF DNA

C. universal oligonucleotides.

FIG. 13 shows

A. a scheme for polymerase chain reaction (PCR) amplification of rat SCFcDNA

B. a scheme for PCR amplification of human SCF cDNA.

FIG. 14 shows

A. sequencing strategy for rat genomic DNA

B. the nucleic acid sequence of rat genomic DNA.

C. the nucleic acid sequence of rat SCF cDNA and amino acid sequence ofrat SCF protein.

FIG. 15 shows

A. the strategy for sequencing human genomic DNA

B. the nucleic acid sequence of human genomic DNA

C. the composite nucleic acid sequence of human SCF cDNA and amino acidsequence of SCF protein.

D. the nucleic acid sequence of genomic DNA and amino acid sequence ofhuman SCF protein, including flanking regions and introns.

FIGS. 16A and B shows the aligned amino acid sequences of human, monkey,dog, mouse, and rat SCF protein.

FIG. 16C shows an elution profile of hSCF¹⁻²⁴⁸ from CHO cells after AspNpeptidase digestion and HPLC.

FIG. 16D shows the nucleotide sequence of the 221 base pair EcoRI-BamHIfragment constructed from oligonucleotides that were used in creatingthe plasmid for human [Met⁻¹] SCF¹⁻¹⁶⁵. Uppercase letters below thenucleotide sequence represent the amino acid sequence. Lowercase lettersabove the nucleotide sequence show nucleotides in the original hSCF¹⁻¹⁸³sequence that were altered to generate codons most frequently used by E.coli.

FIG. 16E shows the 39 base pair BstEII-BamHI fragment used in creatingthe plasmid for human [Met⁻¹] SCF¹⁻¹⁶⁵ with optimized C-terminal codons.

FIG. 17 shows the structure of mammalian cell expression vector V19.8SCF.

FIG. 18 shows the structure of mammalian CHO cell expression vectorpDSVE.1.

FIG. 19 shows the structure of E. coli expression vector pCFM1156.

FIG. 20 shows

A. a radioimmunoassay of mammalian SCF

B. SDS-PAGE of immune-precipitated mammalian SCF.

FIG. 21 shows Western analysis of recombinant human SCF.

FIG. 22 shows Western analysis of recombinant rat SCF.

FIG. 22A shows radioimmune assay determination of SCF in Human Serum.The percent inhibition of ¹²⁵I-human SCF binding produced was determinedfor various doses of an unlabeled standard of CHO HuSCF¹⁻²⁴⁸ (solidcircles); a sample of NHS Lot 500080713 (open circles); and NHS Lot500081015 (solid triangle).

FIG. 23 is a bar graph showing the effect of COS-1 cell-producedrecombinant rat SCF on bone marrow transplantation.

FIG. 24 shows the effect of recombinant rat SCF on curing the macrocyticanemia of Steel mice.

FIG. 25 shows the peripheral white blood cell count (WBC) of Steel micetreated with recombinant rat SCF.

FIG. 26 shows the platelet counts of Steel mice treated with recombinantrat SCF.

FIG. 27 shows the differential WBC count for Steel mice treated withrecombinant rat SCF¹⁻¹⁶⁴ PEG25.

FIG. 28 shows the lymphocyte subsets for Steel mice treated withrecombinant rat SCF¹⁻¹⁶⁴ PEG25.

FIG. 29 shows the effect of recombinant human sequence SCF treatment ofnormal primates in increasing peripheral WBC count.

FIG. 30 shows the effect of recombinant human sequence SCF treatment ofnormal primates in increasing hematocrits and platelet numbers.

FIG. 31 shows photographs of

A. human bone marrow colonies stimulated by recombinant human SCF¹⁻¹⁶²

B. Wright-Giemsa stained cells from colonies in FIG. 31 A.

FIG. 31C shows proliferation of the UT-7 cell line by E. coli derivedSCFs. Open squares are human [Met⁻¹]SCF¹⁻¹⁶⁴, crosses and open diamondsare human [Met⁻¹]SCF¹⁻¹⁶⁵.

FIG. 32 shows SDS-PAGE of S-Sepharose column fractions from chromatogramshown in FIG. 33

A. with reducing agent

B. without reducing agent.

FIG. 33 is a chromatogram of an S-Sepharose column of E. coli derivedrecombinant human SCF.

FIG. 34 shows SDS-PAGE of C₄ column fractions from chromatogram showingFIG. 35

A. with reducing agent

B. without reducing agent.

FIG. 35 is a chromatogram of a C₄ column of E. coli derived recombinanthuman SCF.

FIG. 36 is a chromatogram of a Q-Sepharose column of CHO derivedrecombinant rat SCF.

FIG. 37 is a chromatogram of a C₄ column of CHO derived recombinant ratSCF.

FIG. 38 shows SDS-PAGE of C₄ column fractions from chromatogram shown inFIG. 37.

FIG. 39 shows SDS-PAGE of purified CHO derived recombinant rat SCFbefore and after de-glycosylation.

FIG. 40 shows

A. gel filtration chromatography of recombinant rat pegylated SCF¹⁻¹⁶⁴reaction mixture

B. gel filtration chromatography of recombinant rat SCF¹⁻¹⁶⁴,unmodified.

FIG. 41 shows labelled SCF binding to fresh leukemic blasts.

FIG. 42 shows human SCF cDNA sequence obtained from the HT1080fibrosarcoma cell line.

FIG. 43 shows an autoradiograph from COS-7 cells expressing humanSCF¹⁻²⁴⁸ and CHO cells expressing human SCF¹⁻¹⁶⁴.

FIG. 44 shows human SCF cDNA sequence obtained from the 5637 bladdercarcinoma cell line.

FIG. 45 shows the enhanced survival of irradiated mice after SCFtreatment.

FIG. 46 shows the enhanced survival of irradiated mice after bone marrowtransplantation with 5% of a femur and SCF treatment.

FIG. 47 shows the enhanced survival of irradiated mice after bone marrowtransplantation with 0.1 and 20% of a femur and SCF treatment.

FIG. 48 shows radioprotection effects of rat SCF on survival of miceafter irradiation.

FIG. 49 shows radioprotection effects of rat SCF on survival of miceafter irradiation.

FIG. 50 shows a single coinjection of rrSCF plus G-CSF causes anincrease in circulating neutrophils that is approximately additive ascompared to the rrSCF alone- and G-CSF alone-induced neutrophilia. Thekinetics of rrSCF plus G-CSF-induced neutrophilia reflect the combinedeffect of the differing kinetics of rrSCF-induced neutrophilia peakingat 6 hours and G-CSF-induced neutrophilia peaking at 12 hours.

FIG. 51 shows daily coinjection of rrSCF and G-CSF for one week caused ahighly synergistic increase in circulating neutrophils with a markedlinear increase between day 4 and day 6.

FIG. 52 shows a kinetic study of rrSCF plus G-CSF-induced neutrophiliaafter the seventh daily injection shows that the peak of circulatingneutrophils occurs at 12 hours and reaches a level of 69×10³ PMN/mm³.

FIG. 53 shows in vivo administration of SCF-platelet counts.

FIG. 54 shows dose response of rratSCF-PEG on platelet counts.

FIG. 55 shows effect of 5-FU on platelet levels.

FIG. 56 shows 5-FU effect on ACH+ cells in marrow.

FIG. 57 shows mean platelet volume after 5-FU treatment.

FIG. 58 shows SCF mRNA levels after 5-FU treatment. The data in thisfigure were generated from the same marrow samples collected in FIG. 56.Data points are the values determined from individual mice.

FIG. 59 shows the effects of HuSCF and zidovudine on peripheral bloodBFU-E in normal donors. Light density cells were plated in duplicate inthe presence of (A) 1 U/ml or (B) 4 U/ml of erythropoietin, fourconcentrations of zidovudine (0, 10⁻⁷ M, 10⁻⁶ M and 10⁻⁵ M) and fourconcentrations of HuSCF (0, 10 ng/ml, 100 ng/ml and 1000 ng/ml). Thebars represent the mean±S.E.M. for the duplicate determinations of bothnormal donors. All of the increases for HuSCF are statisticallysignificant (independent t-test, 2-tailed, p<0.01).

FIG. 60 shows the effects of HuSCF and zidovudine on peripheral bloodBFU-E in normal and HIV-infected donors. Light density cells were platedin duplicate in the presence of 1 U/ml of erythropoietin and fourconcentrations of HuSCF (0, 10 ng/ml, 100 ng/ml and 1000 ng/ml). Thebars represent the mean for the duplicate determinations.

FIG. 61 shows alteration of the BFU-E ID₅₀ of zidovudine by HuSCF. The50% inhibitory concentration for BFU-E for each level of HuSCF wascalculated as described in the text. The bars represent the mean for thetwo normal donors.

FIG. 62 shows effects of HuSCF on AZT suppression of bone marrow cultureas measured by BFU-E.

FIG. 63 shows effect of HuSCF on AZT suppression of bone marrow cultureas measured by CFU-GM.

FIG. 64 shows effects of HuSCF on gancyclovir suppression of bone marrowculture as measured by BFU-E.

FIG. 65 shows effect of HuSCF on gancyclovir suppression of bone marrowculture as measured by CFU-GM.

FIG. 66 shows effect of rat SCF alone and in combination with CFU-Snumber in a pre-CFU-S assay.

FIG. 67 shows effect of SCF alone and in combination on the recovery ofhemaglobin.

FIG. 68 shows fluorescence emission spectra of human SCF¹⁻¹⁶⁴. Emissionintensity is shown for CHO cell derived [Met⁻¹]SCF¹⁻¹⁶² (dotted line)and E. coli derived [Met⁻¹]SCF¹⁻¹⁶⁴ (solid line).

FIG. 69 shows circular dichroism of SCF. The far ultraviolet spectra (A)and near ultraviolet spectra (B) are shown for CHO cell-derived[Met⁻¹]SCF¹⁻¹⁶² (dotted line) and E. coli derived [Met⁻¹]SCF¹⁻¹⁶⁴ (solidline).

FIG. 70 shows second derivative infrared spectra of SCF. The secondderivative infrared spectra in the amide I region (1700-1620 cm⁻¹) forE. coli derived [Met⁻¹]SCF¹⁻¹⁶⁴ (A) and CHO cell derived [Met⁻¹SCF¹⁻¹⁶²(B) are shown.

Numerous aspects and advantages of the invention will be apparent tothose skilled in the art upon consideration of the following detaileddescription which provides illustrations of the practice of theinvention in its presently-preferred embodiments.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, novel stem cell factors and DNAsequences coding for all or part of such SCFs are provided. The term“stem cell factor” or “SCF” as used herein refers to naturally-occurringSCF (e.g. natural human SCF) as well as non-naturally occurring (i.e.,different from naturally occurring) polypeptides having amino acidsequences and glycosylation sufficiently duplicative of that ofnaturally-occurring stem cell factor to allow possession of ahematopoietic biological activity of naturally-occurring stem cellfactor. Stem cell factor has the ability to stimulate growth of earlyhematopoietic progenitors which are capable of maturing to erythroid,megakaryocyte, granulocyte, lymphocyte, and macrophage cells. SCFtreatment of mammals results in absolute increases in hematopoieticcells of both myeloid and lymphoid lineages. One of the hallmarkcharacteristics of stem cells is their ability to differentiate intoboth myeloid and lymphoid cells [Weissman, Science, 241, 58-62 (1988)].Treatment of Steel mice (Example 8B) with recombinant rat SCF results inincreases of granulocytes, monocytes, erythrocytes, lymphocytes, andplatelets. Treatment of normal primates with recombinant human SCFresults in increases in myeloid and lymphoid cells (Example 8C).

There is embryonic expression of SCF by cells in the migratory pathwayand homing sites of melanoblasts, germ cells, hematopoietic cells, brainand spinal chord.

Early hematopoietic progenitor cells are enriched in bone marrow frommammals which has been treated with 5-Fluorouracil (5-FU). Thechemotherapeutic drug 5-FU selectively depletes late hematopoieticprogenitors. SCF is active on post 5-FU bone marrow.

The biological activity and pattern of tissue distribution of SCFdemonstrates its central role in embryogenesis and hematopoiesis as wellas its capacity for treatment of various stem cell deficiencies.

The present invention provides DNA sequences which include: theincorporation of codons “preferred” for expression by selectednonmammalian hosts; the provision of sites for cleavage by restrictionendonuclease enzymes; and the provision of additional initial, terminalor intermediate DNA sequences which facilitate construction ofreadily-expressed vectors. The present invention also provides DNAsequences coding for polypeptide analogs or derivatives of SCF whichdiffer from naturally-occurring forms in terms of the identity orlocation of one or more amino acid residues (i.e., deletion analogscontaining less than all of the residues specified for SCF; substitutionanalogs, wherein one or more residues specified are replaced by otherresidues; and addition analogs wherein one or more amino acid residuesis added to a terminal or medial portion of the polypeptide) and whichshare some or all the properties of naturally-occurring forms. Thepresent invention specifically provides DNA sequences encoding the fulllength unprocessed amino acid sequence as well as DNA sequences encodingthe processed form of SCF.

Novel DNA sequences of the invention include sequences useful insecuring expression in procaryotic or eucaryotic host cells ofpolypeptide products having at least a part of the primary structuralconformation and one or more of the biological properties ofnaturally-occurring SCF. DNA sequences of the invention specificallycomprise: (a) DNA sequences set forth in FIGS. 14B, 14C, 15B, 15C, 15D,42 and 44 or their complementary strands; (b) DNA sequences whichhybridize (under hybridization conditions disclosed in Example 3 or morestringent conditions) to the DNA sequences in FIGS. 14B, 14C, 15B, 15C,15D, 42, and 44 or to fragments thereof; and (c) DNA sequences which,but for the degeneracy of the genetic code, would hybridize to the DNAsequences in FIGS. 14B, 14C, 15B, 15C, 15D, 42, and 44. Specificallycomprehended in parts (b) and (c) are genomic DNA sequences encodingallelic variant forms of SCF and/or encoding SCF from other mammalianspecies, and manufactured DNA sequences encoding SCF, fragments of SCF,and analogs of SCF. The DNA sequences may incorporate codonsfacilitating transcription and translation of messenger RNA in microbialhosts. Such manufactured sequences may readily be constructed accordingto the methods of Alton et al., PCT published application WO 83/04053.

According to another aspect of the present invention, the DNA sequencesdescribed herein which encode polypeptides having SCF activity arevaluable for the information which they provide concerning the aminoacid sequence of the mammalian protein which have heretofore beenunavailable. The DNA sequences are also valuable as products useful ineffecting the large scale synthesis of SCF by a variety of recombinanttechniques. Put another way, DNA sequences provided by the invention areuseful in generating new and useful viral and circular plasmid DNAvectors, new and useful transformed and transfected procaryotic andeucaryotic host cells (including bacterial and yeast cells and mammaliancells grown in culture), and new and useful methods for cultured growthof such host cells capable of expression of SCF and its relatedproducts.

DNA sequences of the invention are also suitable materials for use aslabeled probes in isolating human genomic DNA encoding SCF and othergenes for related proteins as well as cDNA and genomic DNA sequences ofother mammalian species. DNA sequences may also be useful in variousalternative methods of protein synthesis (e.g., in insect cells) or ingenetic therapy in humans and other mammals. DNA sequences of theinvention are expected to be useful in developing transgenic mammalianspecies which may serve as eucaryotic “hosts” for production of SCF andSCF products in quantity. See, generally, Palmiter et al., Science 222,809-814 (1983).

The present invention provides purified and isolated naturally-occurringSCF (i.e. purified from nature or manufactured such that the primary,secondary and tertiary conformation, and the glycosylation pattern areidentical to naturally-occurring material) as well as non-naturallyoccurring polypeptides having a primary structural conformation (i.e.,continuous sequence of amino acid residues) and glycosylationsufficiently duplicative of that of naturally occurring stem cell factorto allow possession of a hematopoietic biological activity of naturallyoccurring SCF. Such polypeptides include derivatives and analogs.

In a preferred embodiment, SCF is characterized by being the product ofprocaryotic or eucaryotic host expression (e.g., by bacterial, yeast,higher plant, insect and mammalian cells in culture) of exogenous DNAsequences obtained by genomic or cDNA cloning or by gene synthesis. Thatis, in a preferred embodiment, SCF is “recombinant SCF.” The products ofexpression in typical yeast (e.g., Saccharomyces cerevisiae) orprocaryote (e.g., E. coli) host cells are free of association with anymammalian proteins. The products of expression in vertebrate [e.g.,non-human mammalian (e.g. COS or CHO) and avian] cells are free ofassociation with any human proteins. Depending upon the host employed,polypeptides of the invention may be glycosylated with mammalian orother eucaryotic carbohydrates or may be non-glycosylated. The host cellcan be altered using techniques such as those described in Lee et al. J.Biol. Chem. 264, 13848 (1989) hereby incorporated by reference.Polypeptides of the invention may also include an initial methionineamino acid residue (at position −1).

In addition to naturally-occurring allelic forms of SCF, the presentinvention also embraces other SCF products such as polypeptide analogsof SCF. Such analogs include fragments of SCF. Following the proceduresof the above-noted published application by Alton et al. (WO 83/04053),one can readily design and manufacture genes coding for microbialexpression of polypeptides having primary conformations which differfrom that herein specified for in terms of the identity or location ofone ore more residues (e.g., substitutions, terminal and intermediateadditions and deletions). Alternately, modifications of cDNA and genomicgenes can be readily accomplished by well-known site-directedmutagenesis techniques and employed to generate analogs and derivativesof SCF. Such products share at least one of the biological properties ofSCF but may differ in others. As examples, products of the inventioninclude those which are foreshortened by e.g., deletions; or those whichare more stable to hydrolysis (and, therefore, may have more pronouncedor longer-lasting effects than naturally-occurring); or which have beenaltered to delete or to add one or more potential sites forO-glycosylation and/or N-glycosylation or which have one or morecysteine residues deleted or replaced by, e.g., alanine or serineresidues and are potentially more easily isolated in active form frommicrobial systems; or which have one or more tyrosine residues replacedby phenylalanine and bind more or less readily to target proteins or toreceptors on target cells. Also comprehended are polypeptide fragmentsduplicating only a part of the continuous amino acid sequence orsecondary conformations within SCF, which fragments may possess oneproperty of SCF (e.g., receptor binding) and not others (e.g., earlyhematopoietic cell growth activity). It is noteworthy that activity isnot necessary for any one or more of the products of the invention tohave therapeutic utility [see, Weiland et al., Blut, 44, 173-175 (1982)]or utility in other contexts, such as in assays of SCF antagonism.Competitive antagonists may be quite useful in, for example, cases ofoverproduction of SCF or cases of human leukemias where the malignantcells overexpress receptors for SCF, as indicated by the overexpressionof SCF receptors in leukemic blasts (Example 13).

Of applicability to polypeptide analogs of the invention are reports ofthe immunological property of synthetic peptides which substantiallyduplicate the amino acid sequence extant in naturally-occurringproteins, glycoproteins and nucleoproteins. More specifically,relatively low molecular weight polypeptides have been shown toparticipate in immune reactions which are similar in duration and extentto the immune reactions of physiologically-significant proteins such asviral antigens, polypeptide hormones, and the like. Included among theimmune reactions of such polypeptides is the provocation of theformation of specific antibodies in immunologically-active animals[Lerner et al., Cell, 23, 309-310 (1981); Ross et al., Nature, 294,654-656 (1981); Walter et al., Proc. Natl. Acad. Sci. USA, 77, 5197-5200(1980); Lerner et al., Proc. Natl. Acad. Sci. USA, 78, 4882-4886 (1981);Wong et al., Proc. Natl. Acad. Sci. USA, 79, 5322-5326 (1982); Baron etal., Cell, 28, 395-404 (1982); Dressman et al., Nature, 295, 185-160(1982); and Lerner, Scientific American, 248, 66-74 (1983)]. See, also,Kaiser et al. [Science, 223, 249-255 (1984)] relating to biological andimmunological properties of synthetic peptides which approximately sharesecondary structures of peptide hormones but may not share their primarystructural conformation.

The present invention also includes that class of polypeptides coded forby portions of the DNA complementary to the protein-coding strand of thehuman cDNA or genomic DNA sequences of SCF, i.e., “complementaryinverted proteins” as described by Tramontano et al. [Nucleic Acid Res.,12, 5049-5059 (1984)].

Representative SCF polypeptides of the present invention include but arenot limited to SCF¹⁻¹⁴⁸, SCF¹⁻¹⁶², SCF¹⁻¹⁶⁴, SCF¹⁻¹⁶⁵ and SCF¹⁻¹⁸³ inFIG. 15C; SCF¹⁻¹⁸⁵, SCF¹⁻¹⁸⁸, SCF¹⁻¹⁸⁹ and SCF¹⁻²⁴⁸ in FIG. 42; andSCF¹⁻¹⁵⁷, SCF¹⁻¹⁶⁰, SCF¹⁻¹⁶¹ and SCF¹⁻²²⁰ in FIG. 44.

SCF can be purified using techniques known to those skilled in the art.The subject invention comprises a method of purifying SCF from an SCFcontaining material such as conditioned media or human urine, serum, themethod comprising one or more of steps such as the following: subjectingthe SCF containing material to ion exchange chromatography (eithercation or anion exchange chromatography); subjecting the SCF containingmaterial to reverse phase liquid chromatographic separation involving,for example, an immobilized C₄ to C₆ resin; subjecting the fluid toimmobilized-lectin chromatography, i.e., binding of SCF to theimmobilized lectin, and eluting with the use of a sugar that competesfor this binding. Details in the use of these methods will be apparentfrom the descriptions given in Examples 1, 10, and 11 for thepurification of SCF. The techniques described in Example 2 of the Lai etal. U.S. Pat. No. 4,667,016, hereby incorporated by reference are alsouseful in purifying stem cell factor.

Isoforms of SCF are isolated using standard techniques such as thetechniques set forth in commonly owned U.S. Ser. No. 421,444 entitledErythropoietin Isoforms, filed Oct. 13, 1989, hereby incorporated byreference.

Also comprehended by the invention are pharmaceutical compositionscomprising therapeutically effective amounts of polypeptide products ofthe invention together with suitable diluents, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers useful in SCFtherapy. A “therapeutically effective amount” as used herein refers tothat amount which provides a therapeutic effect for a given conditionand administration regimen. Such compositions are liquids or lyophilizedor otherwise dried formulations and include diluents of various buffercontent (e.g., Tris-HCl., acetate, phosphate), pH and ionic strength,additives such as albumin or gelatin to prevent adsorption to surfaces,detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts),solubilizing agents (e.g., glycerol, polyethylene glycol), anti-oxidants(e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,Thimerosal, benzyl alcohol, parabens), bulking substances or tonicitymodifiers (e.g., lactose, mannitol), covalent attachment of polymerssuch as polyethylene glycol to the protein (described in Example 12below), complexation with metal ions, or incorporation of the materialinto or onto particulate preparations of polymeric compounds such aspolylactic acid, polglycolic acid, hydrogels, etc. or into liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts, or spheroplasts. Such compositions will influencethe physical state, solubility, stability, rate of in vivo release, andrate of in vivo clearance of SCF. The choice of composition will dependon the physical and chemical properties of the protein having SCFactivity. For example, a product derived from a membrane-bound form ofSCF may require a formulation containing detergent. Controlled orsustained release compositions include formulation in lipophilic depots(e.g., fatty acids, waxes, oils). Also comprehended by the invention areparticulate compositions coated with polymers (e.g., poloxamers orpoloxamines) and SCF coupled to antibodies directed againsttissue-specific receptors, ligands or antigens or coupled to ligands oftissue-specific receptors. Other embodiments of the compositions of theinvention incorporate particulate forms, protective coatings, proteaseinhibitors or permeation enhancers for various routes of administration,including parenteral, pulmonary, nasal and oral.

The invention also comprises compositions including one or moreadditional hematopoietic factors such as EPO, G-CSF, GM-CSF, CSF-1,IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IGF-I, or LIF (Leukemic Inhibitory Factor).

Polypeptides of the invention may be “labeled” by association with adetectable marker substance (e.g., radiolabeled with ¹²⁵I orbiotinylated) to provide reagents useful in detection and quantificationof SCF or its receptor bearing cells in solid tissue and fluid samplessuch as blood or urine.

Biotinylated SCF is useful in conjunction with immobilized streptavidinto purge leukemic blasts from bone marrow in autologous bone marrowtransplantation. Biotinylated SCF is useful in conjunction withimmobilized streptavidin to enrich for stem cells in autologous orallogeneic stem cells in autologus or allogeneic bone marrowtransplantation. Toxin conjugates of SCF, such as ricin [Uhr, Prog.Clin. Biol. Res. 288, 403-412 (1988)] diptheria toxid [Moolten, J. Natl.Con. Inst., 55, 473-477 (1975)], and radioisotopes are useful for directanti-neoplastic therapy (Example 13) or as a conditioning regimen forbone marrow transplantation.

Nucleic acid products of the invention are useful when labeled withdetectable markers (such as radiolabels and non-isotopic labels such asbiotin) and employed in hybridization processes to locate the human SCFgene position and/or the position of any related gene family in achromosomal map. They are also useful for identifying human SCF genedisorders at the DNA level and used as gene markers for identifyingneighboring genes and their disorders. The human SCF gene is encoded onchromosome 12, and the murine SCF gene maps to chromosome 10 at the S1locus.

SCF is useful, alone or in combination with other therapy, in thetreatment of a number of hematopoietic disorders. SCF can be used aloneor with one or more additional hematopoietic factors such as EPO, G-CSF,GM-CSF, CSF-1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IGF-I, or LIF in the treatment of hematopoietic disorders.

There is a group of stem cell disorders which are characterized by areduction in functional marrow mass due to toxic, radiant, orimmunologic injury and which may be treatable with SCF. Aplastic anemiais a stem cell disorder in which there is a fatty replacement ofhematopoietic tissue and pancytopenia. SCF enhances hematopoietic tissueand pancytopenia. SCF enhances hematopoietic proliferation and is usefulin treating aplastic anemia (Example 8B). Steel mice are used as a modelof human aplastic anemia [Jones, Exp. Hematol., 11, 571-580 (1983)].Promising results have been obtained with the use of a related cytokine,GM-CSF in the treatment of aplastic anemia [Antin, et al., Blood, 70,129a (1987)]. Paroxysmal nocturnal hemoglobinuria (PNH) is a stem celldisorder characterized by formation of defective platelets andgranulocytes as well as abnormal erythrocytes.

There are many diseases which are treatable with SCF. These include thefollowing: myelofibrosis, myelosclerosis, osteopetrosis, metastaticcarcinoma, acute leukemia, multiple myeloma, Hodgkin's disease,lymphoma, Gaucher's disease, Niemann-Pick disease, Letterer-Siwedisease, refractory erythroblastic anemia, Di Guglielmo syndrome,congestive splenomegaly, Hodgkin's disease, Kala azar, sarcoidosis,primary splenic pancytopenia, miliary tuberculosis, disseminated fungusdisease, Fulminating septicemia, malaria, vitamin B₁₂ and folic aciddeficiency, pyridoxine deficiency, Diamond Blackfan anemia,hypopigmentation disorders such as piebaldism and vitiligo. Theerythroid, megakaryocyte, and granulocytic stimulatory properties of SCFare illustrated in Example 8B and 8C.

Enhancement of growth in non-hematopoietic stem cells such as primordialgerm cells, neural crest derived melanocytes, commissural axonsoriginating from the dorsal spinal cord, crypt cells of the gut,mesonephric and metanephric kidney tubules, and olfactory bulbs is ofbenefit in states where specific tissue damage has occurred to thesesites. SCF is useful for treating neurological damage and is a growthfactor for nerve cells. SCF is useful during in vitro fertilizationprocedures or in treatment of infertility states. SCF is useful fortreating intestinal damage resulting from irradiation or chemotheraphy.

There are stem cell myeloproliferative disorders such as polycythemiavera, chronic myelogenous leukemia, myeloid mataplasia, primarythrombocythemia, and acute leukemias which are treatable with SCF,anti-SCF antibodies, or SCF-toxin conjugates.

There are numerous cases which document the increased proliferation ofleukemic cells to the hematopoietic cell growth factors G-CSF, GM-CSF,and IL-3 [Delwel, et al., Blood, 72, 1944-1949 (1988)]. Since thesuccess of many chemotherapeutic drugs depends on the fact thatneoplastic cells cycle more actively than normal cells, SCF alone or incombination with other factors acts as a growth factor for neoplasticcells and sensitizes them to the toxic effects of chemotherapeuticdrugs. The overexpression of SCF receptors on leukemic blasts is knownin Example 13.

A number of recombinant hematopoietic factors are undergoinginvestigation for their ability to shorten the leukocyte nadir resultingfrom chemotheraphy and radiation regimens. Although these factors arevery useful in this setting, there is an early hematopoietic compartmentwhich is damaged, especially by radiation, and has to be repopulatedbefore these later-acting growth factors can exert their optimal action.The use of SCF alone or in combination with these factors furthershortens or eliminates the leukocyte and platelet nadir resulting fromchemotherapy or radiation treatment. In addition, SCF allows for a doseintensification of the anti-neoplastic or irradiation regime (Example19).

SCF is useful for expanding early hematopoietic progenitors insyngeneic, allogeneic, or autologous bone marrow transplantation. Theuse of hematopoietic growth factors has been shown to decrease the timefor neutrophil recovery after transplantation [Donahue, et al., Nature,321, 872-875 (1986) and Welte et al., J. Exp. Med., 165, 941-948,(1987)]. For bone marrow transplantation, the following three scenariosare used alone or in combination: a donor is treated with SCF alone orin combination with other hematopoietic factors prior to bone marrowaspiration or peripheral blood leucophoresis to increase the number ofcells available for transplantation; the bone marrow is treated in vitroto activate or expand the cell number prior to transplantation; finally,the recipient is treated to enhance engraftment of the donor marrow.

SCF is useful for enhancing the efficiency of gene therapy based ontransfecting (or infecting with a retroviral vector) hematopoietic stemcells. SCF permits culturing and multiplication of the earlyhematopoietic progenitor cells which are to be transfected. The culturecan be done with SCF alone or in combination with IL-6, IL-3, or both.Once tranfected, these cells are then infused in a bone marrowtransplant into patients suffering from genetic disorders. [Lim, Proc.Natl. Acad. Sci, 86, 8892-8896 (1989)]. Examples of genes which areuseful in treating genetic disorders include adenosine deaminase,glucocerebrosidase, hemoglobin, and cystic fibrosis.

SCF is useful for treatment of acquired immune deficiency (AIDS) orsevere combined immunodeficiency states (SCID) alone or in combinationwith other factors such as IL-7 (see Example 14). Illustrative of thiseffect is the ability of SCF therapy to increase the absolute level ofcirculating T-helper (CD4+, OKT₄+) lymphocytes. These cells are theprimary cellular target of human immunodeficiency virus (HIV) leading tothe immunodeficiency state in AIDS patients [Montagnier, in Human T-CellLeukemia/Lymphoma Virus, ed. R. C. Gallo, Cold Spring Harbor, N.Y.,369-379 (1984)]. In addition, SCF is useful for combatting themyelosuppressive effects of anti-HIV drugs such as AZT [Gogu LifeSciences, 45, No. 4 (1989)].

SCF is useful for enhancing hematopoietic recovery after acute bloodloss.

In vivo treatment with SCF is useful as a boost to the immune system forfighting neoplasia (cancer). An example of the therapeutic utility ofdirect immune function enhancement by a recently cloned cytokine (IL-2)is described in Rosenber et al., N. Eng. J. Med., 313 1485 (1987).

The administration of SCF with other agents such as one or more otherhematopoietic factors, is temporally spaced or given together. Priortreatment with SCF enlarges a progenitor population with responds toterminally-acting hematopoietic factors such as G-CSF or EPO. The routeof administration may be intravenous, intraperitoneal sub-cutaneous, orintramuscular.

The subject invention also relates to antibodies specifically bindingstem cell factor. Example 7 below describes the production of polyclonalantibodies. A further embodiment of the invention is monoclonalantibodies specifically binding SCF (see Example 20). In contrast toconventional antibody (polyclonal) preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. Monoclonal antibodies are useful to improve the selectivity andspecificity of diagnostic and analytical assay methods usingantigen-antibody binding. Also, they are used to neutralize or removeSCF from serum. A second advantage of monoclonal antibodies is that theycan be synthesized by hybridoma cells in culture, uncontaminated byother immunoglobulins. Monoclonal antibodies may be prepared fromsupernatants of cultured hybridoma cells or from ascites induced byintraperitoneal inoculation of hybridoma cells into mice. The hybridomatechnique described originally by Köhler and Milstein [Eur. J. Immunol.6, 511-519 (1976)] has been widely applied to produce hybrid cell linesthat secrete high levels of monoclonal antibodies against many specificantigens.

The following examples are offered to more fully illustrate theinvention, but are not to be construed as limiting the scope thereof.

EXAMPLE 1 Purification/Characterization of Stem Cell Factor from BuffaloRat Liver Cell Conditioned Medium

A. In Vitro Biological Assays

1. HPP-CFC Assay

There are a variety of biological activities which can be attributed tothe natural mammalian rat SCF as well as the recombinant rat SCFprotein. One such activity is its effect on early hematopoietic cells.This activity can be measured in a High Proliferative Potential ColonyForming Cell (HPP-CFC) assay [Zsebo, et al., supra (1988)]. Toinvestigate the effects of factors on early hematopoietic cells, theHPP-CFC assay system utilizes mouse bone marrow derived from animals 2days after 5-fluorouracil (5-FU) treatment. The chemotherapeutic drug5-FU selectively depletes late hematopoietic progenitors, allowing fordetection of early progenitor cells and hence factors which act on suchcells. The rat SCF is plated in the presence of CSF-1 or IL-6 insemi-solid agar cultures. The agar cultures contain McCoys completemedium (GIBCO), 20% fetal bovine serum, 0.3% agar, and 2×10⁵ bone marrowcells/ml. The McCoys complete medium contains the following components:1×McCoys medium supplemented with 0.1 mM pyruvate, 0.24×essential aminoacids, 0.24×non-essential amino acids, 0.027% sodium bicarbonate,0.24×vitamins, 0.72 mM glutamine, 25 μg/ml L-serine, and 12 μg/mlL-asparagine. The bone marrow cells are obtained from Balb/c miceinjected i.v. with 150 mg/kg 5-FU. The femurs are harvested 2 days post5-FU treatment of the mice and bone marrow is flushed out. The red bloodcells are lysed with red blood cell lysing reagent (Becton Dickenson)prior to plating. Test substances are plated with the above mixture in30 mm dishes. Fourteen days later the colonies (>1 mm in diameter) whichcontain thousands of cells are scored. This assay was used throughoutthe purification of natural mammalian cell-derived rat SCF.

In a typical assay, rat SCF causes the proliferation of approximately 50HPP-CFC per 200,000 cells plated. The rat SCF has a synergistic activityon 5-FU treated mouse bone marrow cells; HPP-CFC colonies will not formin the presence of single factors but the combination of SCF and CSF-1or SCF and IL-6 is active in this assay.

2. MC/9 Assay

Another useful biological activity of both naturally-derived andrecombinant rat SCF is the ability to cause the proliferation of theIL-4 dependent murine mast cell line, MC/9 (ATCC CRL 8306). MC/9 cellsare cultured with a source of IL-4 according to the ATCC CRL 8306protocol. The medium used in the bioassay is RPMI 1640, 4% fetal bovineserum, 5×10⁻⁵M 2-mercaptoethanol, and 1×glutamine-pen-strep. The MC/9cells proliferate in response to SCF without the requirement for othergrowth factors. This proliferation is measured by first culturing thecells for 24 h without growth factors, plating 5000 cells in each wellof 96 well plates with test sample for 48 h, pulsing for 4 h with 0.5uCi ³H-thymidine (specific activity 20 Ci/mmol), harvesting the solutiononto glass fiber filters, and then measuring specifically-boundradioactivity. This assay was used in the purification of mammalian cellderived rat SCF after the ACA 54 gel filtration step, section C2 of thisExample. Typically SCF caused a 4-10 fold increase in CPM overbackground.

3. CFU-GM

The action of purified mammalian SCF, both naturally-derived andrecombinant, free from interfering colony stimulating factors (CSFs), onnormal undepleted mouse bone marrow has been ascertained. A CFU-GM assay[Broxmeyer et al. Exp. Hematol., 5, 87 (1977)] is used to evaluate theeffect of SCF on normal marrow. Briefly, total bone marrow cells afterlysis of red blood cells are plated in semi-solid agar culturescontaining the test substance. After 10 days, the colonies containingclusters of >40 cells are scored. The agar cultures can be dried downonto glass slides and the morphology of the cells can be determined viaspecific histological stains.

On normal mouse bone marrow, the purified mammalian rat SCF was apluripotential CSF, stimulating the growth of colonies consisting ofimmature cells, neutrophils, macrophages, eosinophils, andmegakaryocytes without the requirement for other factors. From 200,000cells plated, over 100 such colonies grow over a 10 day period. Both ratand human recombinant SCF stimulate the production of erythroid cells incombination with EPO, see Example 9.

B. Conditioned Medium

Buffalo rat liver (BRL) 3A cells, from the American Type CultureCollection (ATCC CRL 1442), were grown on microcarriers in a 20 literperfusion culture system for the production of SCF. This system utilizesa Biolafitte fermenter (Model ICC-20) except for the screens used forretention of microcarriers and the oxygenation tubing. The 75 micronmesh screens are kept free of microcarrier clogging by periodic backflushing achieved through a system of check valves andcomputer-controlled pumps. Each screen alternately acts as medium feedand harvest screen. This oscillating flow pattern ensures that thescreens do not clog. Oxygenation was provided through a coil of siliconetubing (50 feet long, 0.25 inch ID, 0.03 inch wall). The growth mediumused for the culture of BRL 3A cells was Minimal Essential Medium (withEarle's Salts) (GIBCO), 2 mM glutamine, 3 g/L glucose, tryptosephosphate (2.95 g/L), 5% fetal bovine serum and 5% fetal calf serum. Theharvest medium was identical except for the omission of serum. Thereaction contained Cytodex 2 microcarriers (Pharmacia) at aconcentration of 5 g/L and was seeded with 3×10⁹ BRL 3A cells grown inroller bottles and removed by trypsinization. The cells were allowed toattach to and grow on the microcarriers for eight days. Growth mediumwas perfused through the reactor as needed based on glucose consumption.The glucose concentration was maintained at approximately 1.5 g/L. Aftereight days, the reactor was perfused with six volumes of serum freemedium to remove most of the serum (protein concentration<50 ug/ml). Thereactor was then operated batchwise until the glucose concentration fellbelow 2 g/L. From this point onward, the reactor was operated at acontinuous perfusion rate of approximately 10 L/day. The pH of theculture was maintained at 6.9±0.3 by adjusting the CO₂ flow rate. Thedissolved oxygen was maintained higher than 20% of air saturation bysupplementing with pure oxygen as necessary. The temperature wasmaintained at 37±0.5° C.

Approximately 336 liters of serum free conditioned medium was generatedfrom the above system and was used as the starting material for thepurification of natural mammalian cell-derived rat SCF.

C. Purification

All purification work was carried out 4° C. unless indicated otherwise.

1. DEAE-cellulose Anion Exchange Chromatography

Conditioned medium generated by serum-free growth of BRL 3A cells wasclarified by filtration through 0.45μ Sartocapsules (Sartorius). Severaldifferent batches (41 L, 27 L, 39 L, 30.2 L, 37.5 L, and 161 L) wereseparately subjected to concentration, diafiltration/buffer exchange,and DEAE-cellulose anion exchange chromatography, in similar fashion foreach batch. The DEAE-cellulose pools were then combined and processedfurther as one batch in sections C2-5 of this Example. To illustrate,the handling of the 41 L batch was as follows. The filtered conditionedmedium was concentrated to −700 ml using a Millipore Pellicon tangentialflow ultrafiltration apparatus with four 10,000 molecular weight cutoffpolysulfone membrane cassettes (20 ft² total membrane area; pump rate−1095 ml/min and filtration rate 250-315 ml/min). Diafiltration/bufferexchange, in preparation for anion exchange chromatography was thenaccomplished by adding 500 ml of 50 mM Tris-HCl, pH 7.8 to theconcentrate, reconcentrating to 500 ml using the tangential flowultrafiltration apparatus, and repeating this six additional times. Theconcentrated/diafiltered preparation was finally recovered in a volumeof 700 ml. The preparation was applied to a DEAE-cellulose anionexchange column (5×20.4 cm; Whatman DE-52 resin) which had beenequilibrated with the 50 mM Tris-HCl, pH 7.8 buffer. After sampleapplication, the column was washed with 2050 ml of the Tris-HCl buffer,and a salt gradient (0-300 mM NaCl in the Tris-HCl buffer, 4 L totalvolume) was applied. Fractions of 15 ml were collected at a flow rate of167 ml/h. The chromatography is shown in FIG. 1. HPP-CFC colony numberrefers to biological activity in the HPP-CFC assay; 100 μl from theindicated fractions was assayed. Fractions collected during the sampleapplication and wash are not shown in the Figure; no biological activitywas detected in these fractions.

The behavior of all conditioned media batches subjected to theconcentration, diafiltration/buffer exchange, and anion exchangechromatography was similar. Protein concentrations for the batches,determined by the method of Bradford [Anal. Biochem. 72, 248-254 (1976)]with bovine serum albumin as standard were in the range 30-50 μg/ml. Thetotal volume of conditioned medium utilized for this preparation wasabout 336 L.

2. ACA 54 Gel Filtration Chromatography

Fractions having biological activity from the DEAE-cellulose columns runfor each of the six conditioned media batches referred to above (forexample, fractions 87-114 for the run shown in FIG. 1) were combined(total volume 2900 ml) and concentrated to a final volume of 74 ml withthe use of Amicon stirred cells and YM10 membranes. This material wasapplied to an ACA 54 (LKB) gel filtration column (FIG. 2) equilibratedin 50 mM Tris-HCl, 50 mM NaCl, pH 7.4. Fractions of 14 ml were collectedat a flow rate of 70 ml/h. Due to inhibitory factors co-eluting with theactive fractions, the peak of activity (HPP-CFC colony number) appearssplit; however, based on previous chromatograms, the activity co-eluteswith the major protein peak and therefore one pool of the fractions wasmade.

3. Wheat Germ Agglutinin-Agarose Chromatography

Fractions 70-112 from the ACA 54 gel filtration column were pooled (500ml). The pool was divided in half and each half subjected tochromatography using a wheat germ agglutinin-agarose column (5×24.5 cm;resin from E-Y Laboratories, San Mateo, Calif.; wheat germ agglutininrecognizes certain carbohydrate structures) equilibrated in 20 mMTris-HCl, 500 mM NaCl, pH 7.4. After the sample applications, the columnwas washed with about 2200 ml of the column buffer, and elution of boundmaterial was then accomplished by applying a solution of 350 mMN-acetyl-D-glucosamine dissolved in the column buffer, beginning atfraction −210 in FIG. 3. Fractions of 13.25 ml were collected at a flowrate of 122 ml/hr. One of the chromatographic runs is shown in FIG. 3.Portions of the fractions to be assayed were dialyzed againstphosphate-buffered saline; 5 ul of the dialyzed materials were placedinto the MC/9 assay (cpm values in FIG. 3) and 10 μl into the HPP-CFCassay (colony number values in FIG. 3). It can be seen that the activematerial bound to the column and was eluted with theN-acetyl-D-glucosamine, whereas much of the contaminating materialpassed through the column during sample application and wash.

4. S-Sepharose Fast Flow Cation Exchange Chromatography

Fractions 211-225 from the wheat germ agglutinin-agarose chromatographyshown in FIG. 3 and equivalent fractions from the second run were pooled(375 ml) and dialyzed against 25 mM sodium formate, pH 4.2. To minimizethe time of exposure to low pH, the dialysis was done over a period of 8h, against 5 L of buffer, with four changes being made during the 8 hperiod. At the end of this dialysis period, the sample volume was 480 mland the pH and conductivity of the sample were close to those of thedialysis buffer. Precipitated material appeared in the sample duringdialysis. This was removed by centrifugation at 22,000 × g for 30 min,and the supernatant from the centrifuged sample was applied to aS-Sepharose Fast Flow cation exchange column (3.3×10.25 cm; resin fromPharmacia) which had been equilibrated in the sodium formate buffer.Flow rate was 465 ml/h and fractions of 14.2 ml were collected. Aftersample application, the column was washed with 240 ml of column bufferand elution of bound material was carried out by applying a gradient of0-750 mM NaCl (NaCl dissolved in column buffer; total gradient volume2200 ml), beginning at fraction ˜45 in FIG. 4. The elution profile isshown in FIG. 4. Collected fractions were adjusted to pH 7-7.4 byaddition of 200 μl of 0.97 M Tris base. The cpm in FIG. 4 again refer tothe results obtained in the MC/9 biological assay; portions of theindicated fractions were dialyzed against phosphate-buffered saline, and20 μl placed into the assay. It can be seen in FIG. 4 that the majorityof biologically active material passed through the column unbound,whereas much of the contaminating material bound and was eluted in thesalt gradient.

5. Chromatography Using Silica-Bound Hydrocarbon Resin

Fractions 4-40 from the S-Sepharose column of FIG. 4 were pooled (540ml). 450 ml of the pool was combined with an equal volume of buffer B(100 mM ammonium acetate, pH 6:isopropanol; 25:75) and applied at a flowrate of 540 ml/h to a C₄ column (Vydac Proteins C₄; 2.4×2 cm)equilibrated with buffer A (60 mM ammonium acetate, pH 6:isopropanol;62.5:37.5). After sample application, the flow rate was reduced to 154ml/h and the column was washed with 200 ml of buffer A. A lineargradient from buffer A to buffer B (total volume 140 ml) was thenapplied, and fractions of 9.1 ml were collected. Portions of the poolfrom S-Sepharose chromatography, the C₄ column starting sample,runthrough pool, and wash pool were brought to 40 μg/ml bovine serumalbumin by addition of an appropriate volume of a 1 mg/ml stocksolution, and dialyzed against phosphate-buffered saline in preparationfor biological assay. Similarly, 40 μl aliquots of the gradientfractions were combined with 360 μl of phosphate-buffered salinecontaining 16 μg bovine serum albumin, and this was followed by dialysisagainst phosphate-buffered saline in preparation for biological assay.These various fractions were assayed by the MC/9 assay (6.3 μl aliquotsof the prepared gradient fractions; cpm in FIG. 5). The assay resultsalso indicated that about 75% of the recovered activity was in therunthrough and wash fractions, and 25% in the gradient fractionsindicated in FIG. 5. SDS-PAGE [Laemmli, Nature, 227, 680-685 (1970);stacking gels contained 4% (w/v) acrylamide and separating gelscontaining 12.5% (w/v) acrylamide] of aliquots of various fractions isshown in FIG. 6. For the gel shown, sample aliquots were dried undervacuum and then redissolved using 20 μl sample treatment buffer(nonreducing, i.e., without 2-mercaptoethanol) and boiled for 5 minprior to loading onto the gel. Lanes A and B represent column startingmaterial (75 μl out of 890 ml) and column runthrough (75 μl out of 880ml), respectively; the numbered marks at the left of the Figurerepresent migration positions (reduced) of markers having molecularweights of 10³ times the indicated numbers, where the markers arephosphorylase b (M_(r) of 97,400), bovine serum albumin (M_(r) of66,200), ovalbumin (M_(r) of 42,700), carbonic anhydrase (M_(r) of31,000), soybean trypsin inhibitor (M_(r) of 21,500) and lysozyme (M_(r)of 14,400); lanes 4-9 represent the corresponding fractions collectedduring application of the gradient (60 μl out of 9.1 ml). The gel wassilver-stained [Morrissey, Anal. Biochem., 117, 307-310 (1981)]. It canbe seen by comparing lanes A and B that the majority of stainablematerial passes through the column. The stained material in fractions4-6 in the regions just above and below the M_(r) 31,000 standardposition coincides with the biological activity detected in the gradientfractions (FIG. 5) and represents the biologically active material. Itshould be noted that this material is visualized in lanes 4-6, but notin lanes A and/or B, because a much larger proportions of the totalvolume (0.66% of the total for fractions 4-6 versus 0.0084% of the totalfor lanes A and B) was loaded for the former. Fractions 4-6 from thiscolumn were pooled.

As mentioned above, roughly 75% of the recovered activity ran throughthe C₄ column of FIG. 5. This material was rechromatographed using C₄resin essentially as described above, except that a larger column(1.4×7.8 cm) and slower flow rate (50-60 ml/h throughout) were used.Roughly 50% of recovered activity was in the runthrough, and 50% ingradient fractions showing similar appearance on SDS-PAGE to that of theactive gradient fractions in FIG. 6. Active fractions were pooled (29ml).

An analytical C₄ column was also performed essentially as stated aboveand the fractions were assayed in both bioassays. As indicated in FIG. 7of the fractions from this analytical column, both the MC/9 and HPP-CFCbioactivities co-elute. SDS-PAGE analysis (FIG. 8) reveals the presenceof the M_(r) ˜31,000 protein in the column fractions which containbiological activity in both assays.

Active material in the second (relatively minor) activity peak seen inS-Sepharose chromatography (e.g. FIG. 4, fractions 62-72, earlyfractions in the salt gradient) has also been purified by C₄chromatography. It exhibited the same behavior on SDS-PAGE and had thesame N-terminal amino acid sequence (see Example 2D) as the materialobtained by C₄ chromatography of the S-Sepharose runthrough fractions.

6. Purification Summary

A summary of the purification steps described in 1-5 above is given inTable 2.

TABLE 2 Summary of Purification of Mammalian SCF Total Step Volume (ml)Protein (mg)⁵ Conditioned medium 335,700 13,475 DEAE cellulose¹  2,9002,164 ACA-54    550 1,513 Wheat germ agglutinin-agarose²    375 431S-Sepharose    540⁴ 10 C₄ resin³     57.3 0.25-0.40⁶ ¹Values givenrepresent sums of the values for the different batches described in thetext. ²As described above in this Example, precipitated material whichappeared during dialysis of this sample in preparation for S-Sepharosechromatography was removed by centrifugation. The sample aftercentrifugation (480 ml) contained 264 mg of total protein. ³Combinationof the active gradient fractions from the two C₄ columns run in sequenceas described. ⁴Only 450 ml of this material was used for the followingstep (this Example, above). ⁵Determined by the method of Bradford(supra, 1976) except where indicated otherwise. ⁶Estimate, based onintensity of silver-staining after SDS-PAGE, and on amino acidcomposition analysis as described in section K of Example 2.

D. SDS-PAGE and Glycosidase Treatments

SDS-PAGE of pooled gradient fractions from the two large scale C₄ columnruns are shown in FIG. 9. Sixty μl of the pool for the first C₄ columnwas loaded (lane 1), and 40 μl of the pool for the second C₄ column(lane 2). These gel lanes were silver-stained. Molecular weight markerswere as described for FIG. 6. As mentioned, the diffusely-migratingmaterial above and below the M_(r) 31,000 marker position represents thebiologically active material; the apparent heterogeneity is largely dueto heterogeneity in glycosylation.

To characterize the glycosylation, purified material was iodinated with¹²⁵I, treated with a variety of glycosidases, and analyzed by SDS-PAGE(reducing conditions) with autoradiography. Results are shown in FIG. 9.Lanes 3 and 9, ¹²⁵I-labeled material without any glycosidase treatment.Lanes 4-8 represent ¹²⁵I-labeled material treated with glycosidases, asfollows. Lane 4, neuraminidase. Lane 5, neuraminidase and O-glycanase.Lane 6, N-glycanase. Lane 7, neuraminidase and N-glycanase. Lane 8,neuraminidase, O-glycanase, and N-glycanase. Conditions were 5 mM3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 33 mM2-mercaptoethanol, 10 mM Tris-HCl, pH 7-7.2, for 3 h at 37° C.Neuraminidase (from Arthrobacter ureafaciens; Calbiochem) was used at0.23 units/ml final concentration. O-Glycanase (Genzyme;endo-alpha-N-acetyl-galactosaminidase) was used at 45 milliunits/ml.N-Glycanase (Genzyme; peptide:N-glycosidase F;peptide-N⁴[N-acetyl-beta-glucosaminyl]asparagine amidase) was used at 10units/ml.

Similar results to those of FIG. 9 were obtained upon treatment ofunlabeled purified SCF with glycosidases, and visualization of productsby silver-staining after SDS-PAGE.

Where appropriate, various control incubations were carried out. Theseincluded: incubation in appropriate buffer, but without glycosidases, toverify that results were due to the glycosidase preparations added;incubation with glycosylated proteins (e.g. glycosylated recombinanthuman erythropoietin) known to be substrates for the glycosidases, toverify that the glycosidase enzymes used were active; and incubationwith glycosidases but no substrate, to verify that the glycosidases werenot themselves contributing to or obscuring the visualized gel bands.

Glycosidase treatments were also carried out withendo-beta-N-acetylglucosamidase F (endo F; NEN Dupont) and withendo-beta-N-acetylglucosaminidase H (endo H; NEN Dupont), again withappropriate control incubations. Conditions of treatment with endo Fwere: boiling 3 min in the presence of 1% (w/v) SDS, 100 mM2-mercaptoethanol, 100 mM EDTA, 320 mM sodium phosphate, pH 6, followedby 3-fold dilution with the inclusion of Nonidet P-40 (1.17%, v/v, finalconcentration), sodium phosphate (200 mM, final concentration), and endoF (7 units/ml, final concentration). Conditions of endo H treatment weresimilar except that SDS concentration was 0.5% (w/v) and endo H was usedat a concentration of 1 μg/ml. The results with endo F were the same asthose with N-glycanase, whereas endo H had no effect on the purified SCFmaterial.

A number of conclusions can be drawn from the glyosidase experimentsdescribed above. The various treatments with N-glycanase [which removesboth complex and high-mannose N-linked carbohydrate (Tarentino et al.,Biochemistry 24, 4665-4671) (1985)], endo F [which acts similarly toN-glycanase (Elder and Alexander, Proc. Natl. Acad. Sci. U.S.A. 79,4540-4544 (1982)], endo H [which removes high-mannose and certain hybridtype N-linked carbohydrate (Tarentino et al., Methods Enzymol. 50C,574-580 (1978)], neuraminidase (which removes sialic acid residues), andO-glycanase [which removes certain O-linked carbohydrates (Lambin etal., Biochem. Soc. Trans. 12, 599-600 (1984)], suggest that: bothN-linked and O-linked carbohydrates are present; most of the N-linkedcarbohydrate is of the complex type; and sialic acid is present, with atleast some of it being part of the O-linked moieties. Some informationabout possible sites of N-linkage can be obtained from amino acidsequence data (Example 2). The fact that treatment with N-glycanase,endo F, and N-glycanase/neuraminidase can convert the heterogeneousmaterial apparent by SDS-PAGE to faster-migrating forms which are muchmore homogeneous is consistent with the conclusion that all of thematerial represents the same polypeptide, with the heterogeneity beingcaused by heterogeneity in glycosylation. It is also noteworthy that thesmallest forms obtained by the combined treatments with the variousglycosidases are in the range of M_(r) 18,000-20,000, relative to themolecular weight markers used in the SDS-PAGE.

Confirmation that the diffusely-migrating material around the M_(r)31,000 position on SDS-PAGE represents biologically active material allhaving the same basic polypeptide chain is given by the fact that aminoacid sequence data derived from material migrating in this region (e.g.,after electrophoretic transfer and cyanogen bromide treatment; Example2) matches that demonstrated for the isolated gene whose expression byrecombinant DNA means leads to biologically-active material (Example 4).

EXAMPLE 2 Amino Acid Sequence Analysis of Mammalian SCF

A. Reverse-phase High Performance Liquid Chromatography (HPLC) ofPurified Protein

Approximately 5 μg of SCF purified as in Example 1 (concentration=0.117mg/ml) was subjected to reverse-phase HPLC using a C₄ narrowbore column(Vydac, 300 Å widebore, 2 mm×15 cm). The protein was eluted with alinear gradient from 97% mobile phase A (0.1% trifluoroacetic acid)/3%mobile phase B (90% acetonitrile in 0.1% trifluoroacetic acid) to 30%mobile phase A/70% mobile phase B in 70 min followed by isocraticelution for another 10 min at a flow rate of 0.2 ml per min. Aftersubtraction of a buffer blank chromatogram, the SCF was apparent as asingle symmetrical peak at a retention time of 70.05 min as shown inFIG. 10. No major contaminating protein peaks could be detected underthese conditions.

B. Sequencing of Electrophoretically-Transferred Protein Bands

SCF purified as in Example 1 (0.5-1.0 nmol) was treated as follows withN-glycanase, an enzyme which specifically cleaves the Asn-linkedcarbohydrate moieties covalently attached to proteins (see Example 1D).Six ml of the pooled material from fractions 4-6 of the C₄ column ofFIG. 5 was dried under vacuum. Then 150 μl of 14.25 mM CHAPS, 100 mM2-mercaptoethanol, 335 mM sodium phosphate, pH 8.6 was added andincubation carried out for 95 min at 37° C. Next 300 μl of 74 mM sodiumphosphate, 15 units/ml N-glycanase, pH 8.6 was added and incubationcontinued for 19 h. The sample was then run on a 9-18%SDS-polyacrylamide gradient gel (0.7 mm thickness, 20×20 cm). Proteinbands in the gel were electrophoretically transferred ontopolyvinyldifluoride (PVDF, Millipore Corp.) using 10 mM Caps buffer (pH10.5) at a constant current of 0.5 Amp for 1 h [Matsudaira, J. Biol.Chem., 261, 10035-10038 (1987)]. The transferred protein bands werevisualized by Coomassie Blue staining. Bands were present at M_(r)˜29,000-33,000 and M_(r) ˜26,000, i.e., the deglycosylation was onlypartial (refer to Example 1D, FIG. 9); the former band representsundigested material and the latter represents material from whichN-linked carbohydrate is removed. The bands were cut out and directlyloaded (40% for M_(r) 29,000-33,000 protein and 80% for M_(r) 26,000protein) onto a protein sequencer (Applied Biosystems Inc., model 477).Protein sequence analysis was performed using programs supplied by themanufacturer [Hewick et al., J. Biol. Chem., 256 7990-7997 (1981)] andthe released phenylthiohydantoinyl amino acids were analyzed on-lineusing microbore C₁₈ reverse-phase HPLC. Both bands gave no signals for20-28 sequencing cycles, suggesting that both were unsequenceable bymethodology using Edman chemistry. The background level on eachsequencing run was between 1-17 pmol which was far below the proteinamount present in the bands. These data suggested that protein in thebands was N-terminally blocked.

C. In situ CNBr Cleavage of Electrophoretically-Transferred Protein andSequencing

To confirm that the protein was in fact blocked, the membranes wereremoved from the sequencer (part B) and in situ cyanogen bromide (CNBr)cleavage of the blotted bands was carried out [CNBr (5%, w/v) in 70%formic acid for 1 h at 45° C.] followed by drying and sequence analysis.Strong sequence signals were detected, representing internal peptidesobtained from methionyl peptide bond cleavage by CNBr.

Both bands yielded identical mixed sequence signals listed below for thefirst five cycles.

Amino Acids Identified Cycle 1: Asp; Glu; Val; Ile; Leu Cycle 2: Asp;Thr; Glu; Ala; Pro; Val Cycle 3: Asn; Ser; His; Pro; Leu Cycle 4: Asp;Asn; Ala; Pro; Leu Cycle 5: Ser; Tyr; Pro

Both bands also yielded similar signals up to 20 cycles. The initialyields were 40-115 pmol for the M_(r) 26,000 band and 40-150 pmol forthe M_(r) 29,000-33,000 band. These values are comparable to theoriginal molar amounts of protein loaded onto the sequencer. The resultsconfirmed that protein bands corresponding to SCF contained a blockedN-terminus. Procedures used to obtain useful sequence information forN-terminally blocked proteins include: (a) deblocking the N-terminus(see section D); and (b) generating peptides by internal cleavages byCNBr (see Section E), by trypsin (see Section F), and by Staphylococcusaureus (strain V-8) protease (Glu-C) (see Section G). Sequence analysiscan proceed after the blocked N-terminal amino acid is removed or thepeptide fragments are isolated. Examples are described in detail below.

D. Sequence Analysis of BRL Stem Cell Factor Treated with PyroglutamicAcid Aminopeptidase

The chemical nature of the blockage moiety present at the amino terminusof SCF was difficult to predict. Blockage can be post-translational invivo [F. Wold, Ann. Rev. Biochem., 50, 783-814 (1981)] or may occur invitro during purification. Two post-translational modifications are mostcommonly observed. Acetylation of certain N-terminal amino acids such asAla, Ser, etc. can occur, catalyzed by N-α-acetyl transferase. This canbe confirmed by isolation and mass spectrometric analysis of anN-terminally blocked peptide. If the amino terminus of a protein inglutamine, deamidation of its gamma-amide can occur. Cyclizationinvolving the gamma-carboxylate and the free N-terminus can then occurto yield pyroglutamate. To detect pyroglutamate, the enzymepyroglutamate aminopeptidase can be used. This enzyme removes thepyroglutamate residue, leaving a free amino terminus starting at thesecond amino acid. Edman chemistry can then be used for sequencing.

SCF (purified as in Example 1; 400 pmol) in 50 mM sodium phosphatebuffer (pH 7.6 containing dithiothreitol and EDTA) was incubated with1.5 units of calf liver pyroglutamic acid aminopeptidase (pE-AP) for 16h at 37° C. After reaction the mixture was directly loaded onto theprotein sequencer. A major sequence could be identified through 46cycles. The initial yield was about 40% and repetitive yield was 94.2%.The N-terminal sequence of SCF including the N-terminal pyroglutamicacid is:

pE-AP cleavage site        ↓                                 10pyroGlu-Glu-Ile-Cys-Arg-Asn-Pro-Val-Thr-Asp-Asn-Val-Lys-Asp-Ile-Thr-Lys-         20                                      30Leu-Val-Ala-Asn-Leu-Pro-Asn-Asp-Tyr-Met-Ile-Thr-Leu-Asn-Tyr-Val-                         40Ala-Gly-Met-Asp-Val-Leu-Pro-Ser-His-xxx-Trp-Leu-Arg-Asp-.........              xxx, not assigned at position 43

These results indicated that SCF contains pyroglutamic acid as itsN-terminus.

E. Isolation and Sequence Analysis of CNBr Peptides

SCF purified as in Example 1 (20-28 μg; 1.0-1.5 mmol) was treated withN-glycanase as described in Example 1. Conversion to the M_(r) 26,000material was complete in this case. The sample was dried and digestedwith CNBr in 70% formic acid (5%) for 18 h at room temperature. Thedigest was diluted with water, dried, and redissolved in 0.1%trifluoroacetic acid. CNBr peptides were separated by reverse-phase HPLCusing a C₄ narrowbore column and elution conditions identical to thosedescribed in Section A of this Example. Several major peptide fractionswere isolated and sequenced, and the results are summarized in thefollowing:

Retention Peptide Time (min) Sequence⁴ CB-4 15.5 L-P-P--- CB-6¹ 22.1 a.I-T-L-N-Y-V-A-G-(M) b. V-A-S-D-T-S-D-C-V-L-S-₋-₋-L-G-P-E-K-D-S-R-V-S-V-(₋)-K---- CB-8 28.0 D-V-L-P-S-H-C-W-L-R-D-(M) CB-10 30.1(containing sequence of CB-8) CB-15² 43.0E-E-N-A-P-K-N-V-K-E-S-L-K-K-P-T-R-(N)-F-T-P-E-E-F-F-S-I-F-D³-R-S-I-D-A------ CB-14 37.3 and CB-16 Both peptidescontain identical sequence to CB-15 ¹Amino acids were not detected atpositions 12, 13 and 25. Peptide b was not sequenced to the end. ²(N) inCB-15 was not detected; it was inferred based on the potential N-linkedglycosylation site. The peptide was not sequenced to the end.³Designates site where Asn may have been converted into Asp uponN-glycanase removal of N-linked sugar. ⁴Single letter code was used: A,Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L,Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W,Trp; and Y, Tyr.

F. Isolation and Sequencing of BRL Stem Cell Factor Tryptic Fragments

SCF purified as in Example 1 (20 μg in 150 μl 0.1 M ammoniumbicarbonate) was digested with 1 μg of trypsin at 37° C. for 3.5 h. Thedigest was immediately run on reverse-phase narrow bore C₄ HPLC usingelution conditions identical to those described in Section A of thisExample. All eluted peptide peaks had retention times different fromthat of undesignated SCF (Section A). The sequence analyses of theisolated peptides are shown below:

Retention Time Peptide (min) Sequence T-1  7.1 E-S-L-K-K-P-E-T-R T-2¹28.1 V-S-V-(₋)-K T-3 32.4 I-V-D-D-L-V-A-A-M-E-E-N-A-P-K T-4² 40.0N-F-T-P-E-E-F-F-S-I-F-(₋)-R T-5³ 46.4 a.L-V-A-N-L-P-N-D-Y-M-I-T-L-N-Y-V-A-G- M-D-V-L-P-S-H-C-W-L-R b.S-I-D-A-F-K-D-F-M-V-A-S-D-T-S-D-C-V- L-S-(₋)-(₋)-L-G---- T-7⁴ 72.8E-S-L-K-K-P-E-T-R-(N)-F-T-P-E-E-F-F- S-I-F-(₋)-R T-8 73.6E-S-L-K-K-P-E-T-R-N-F-T-P-E-E-F-F-S-I- F-D-R ¹Amino acid at position 4was not assigned. ²Amino acid at position 12 was not assigned. ³Aminoacids at positions 20 and 21 in 6 of peptide T-5 were not identified;they were tentatively assigned as O-linked sugar attachment sites.⁴Amino acid at position 10 was not detected; it was inferred as Asnbased on the potential N-linked glycosylation site. Amino acid atposition 21 was not detected.

G. Isolation and Sequencing of BRL Stem Cell Factor Peptides after S.aureus Glu-C Protease Cleavage

SCF purified as in Example 1 (20 μg in 150 μl 0.1 M ammoniumbicarbonate) was subjected to Glu-C protease cleavage at aprotease-to-substrate ratio of 1:20. The digestion was accomplished at37° C. for 18 h. The digest was immediately separated by reverse-phasenarrowbore C₄ HPLC. Five major peptide fractions were collected andsequenced as described below:

Retention Peptides Time (min) Sequence S-1  5.1 N-A-P-K-N-V-K-E S-2¹27.7 S-R-V-S-V-(₋)-K-P-F-M-L-P-P-V-A-(A) S-3² 46.3 No sequence detectedS-5³ 71.0 S-L-K-K-P-E-T-R-N-F-T-P-E-E-F-F-S-I-F-(N)-R-S-I-D-A-F-K-D-F-M-V-A-S-D S-6³ 72.6S-L-K-K-P-E-T-R-N-F-T-P-E-E-F-F-S-I-F-(N)-R-S-I-D-A-F-K-D-F-M-V-A-S-D-T-S-D ¹Amino acid at position 6 of S-2peptide was not assigned; this could be an O-linked sugar attachmentsite. The Ala at position 16 of S-2 peptide was detected in low yield.²Peptide S-3 could be the N-terminally blocked peptide derived from theN-terminus of SCF. ³N in parentheses was assigned as a potentialN-linked sugar attachment site.

H. Sequence Analysis of BRL Stem Cell Factor after BNPS-skatole Cleavage

SCF (2 μg) in 10 mM ammonium bicarbonate was dried to completeness byvacuum centrifugation and then redissolved in 100 ul of glacial aceticacid. A 10-20 fold molar excess of BNPS-skatole was added to thesolution and the mixture was incubated at 50° C. for 60 min. Thereaction mixture was then dried by vacuum centrifugation. The driedresidue was extracted with 100 μl of water and again with 50 μl ofwater. The combined extracts were then subjected to sequence analysis asdescribed above. The following sequence was detected:

1                                    10Leu-Arg-Asp-Met-Val-Thr-His-Leu-Ser-Val-Ser-Leu-Thr-Thr-Leu-Leu-             20                                        30Asp-Lys-Phe-Ser-Asn-Ile-Ser-Glu-Gly-Leu-Ser-(Asn)-Tyr-Ser-Ile-Ile-                             40 Asp-Lys-Leu-Gly-Lys-Ile-Val-Asp----

Position 28 was not positively assigned; it was assigned as Asn based onthe potential N-linked glycosylation site.

I. C-Terminal Amino Acid Determination of BRL Stem Cell Factor

An aliquot of SCF protein (500 pmol) was buffer-exchanged into 10 mMsodium acetate, pH 4.0 (final volume of 90 μl) and Brij-35 was added to0.05% (w/v). A 5 μl aliquot was taken for quantiation of protein. Fortyμl of the sample was diluted to 100 μl with the buffer described above.Carboxypeptidase P (from Penicillium janthinellum) was added at anenzyme-to-substrate ratio of 1:200. The digestion proceeded at 25° C.and 20 μl aliquots were taken at 0, 15, 30, 60 and 120 min. Thedigestion was terminated at each time point by adding trifluoroaceticacid to a final concentration of 5%. The samples were dried and thereleased amino acids were derivatized by reaction with Dabsyl chloride(dimethylaminoazobenzenesulfonyl chloride) in 0.2 M NaHCO₃ (pH 9.0) at70° C. for 12 min [Chang et al., Methods Enzymol., 90, 41-48 (1983)].The derivatized amino acids (one-sixth of each sample) were analyzed bynarrowbore reverse-phase HPLC with a modification of the procedure ofChang et al. [Techniques in Protein Chemistry, T, Hugli ed., Acad.Press, NY (1989), pp. 305-311]. Quantitative composition results at eachtime point were obtained by comparison to derivatized amino acidstandards (1 pmol). At 0 time, contaminating glycine was detected.Alanine was the only amino acid that increased with incubation time.After 2 h incubation, Ala was detected at a total amount of 25 pmol,equivalent to 0.66 mole of Ala released per mole of protein. This resultindicated that the natural mammalian SCF molecule contains Ala as itscarboxyl terminus, consistent with the sequence analysis of a C-terminalpeptide, S-2, which contains C-terminal Ala. This conclusion is alsoconsistent with the known specificity of carboxypeptidase P [Lu et al.,J. Chromatog. 447, 351-364 (1988)]. For example, cleavage ceases if thesequence Pro-Val is encountered. Peptide S-2 has the sequenceS-R-V-S-V-(T)-K-P-F-M-L-P-P-V-A-(A) and was deduced to be the C-terminalpeptide of SCF (see Section J in this Example). The C-terminal sequenceof ---P-V-A-(A) restricts the protease cleavage to alanine only. Theamino acid composition of peptide S-2 indicates the presence of 1 Thr, 2Ser, 3 Pro, 2 Ala, 3 Val, 1 Met, 1 Leu, 1 Phe, 1 Lys, and 1 Arg,totalling 16 residues. The detection of 2 Ala residues indicates thatthere may be two Ala residues at the C-terminus of this peptide (seetable in Section G). Thus the BRL SCF terminates at Ala 164 or Ala 165.

J. Sequence of SCF

By combining the results obtained from sequence analysis of (1) intactstem cell factor after removing its N-terminal pyroglutamic acid, (2)the CNBr peptides, (3) the trypsin peptides, and (4) the Glu-C peptidasefragments, an N-terminal sequence and a C-terminal sequence was deduced(FIG. 11). The N-terminal sequence starts at pyroglutamic acid and endsat Met-48. The C-terminal sequence contains 84/85 amino acids (position82 to 164/165). The sequence from position 49 to 81 was not detected inany of the peptides isolated. However, a sequence was detected for alarge peptide after BNPS-skatole cleavage of BRL SCF as described inSection H of this Example. From these additional data, as well as DNAsequence obtained from rat SCF (Example 3) the N- and C-terminalsequences can be aligned and the overall sequence delineated as shown inFIG. 11. The N-terminus of the molecule is pyroglutamic acid and theC-terminus is alanine as confirmed by pyroglutamate aminopeptidasedigestion and carboxypeptidase P digestion, respectively.

From the sequence data, it is concluded that Asn-72 is glycosylated;Asn-109 and Asn-120 are probably glycosylated in some molecules but notin others. Asn-65 could be detected during sequence analysis andtherefore may only be partially glycosylated, if at all. Ser-142,Thr-143 and Thr-155, predicted from DNA sequence, could not be detectedduring amino acid sequence analysis and therefore could be sites ofO-linked carbohydrate attachment. These potential carbohydrateattachment sites are indicated in FIG. 11; N-linked carbohydrate isindicated by solid bold lettering; O-linked carbohydrate is indicated byopen bold lettering.

Amino Acid Compositional Analysis of BRL Stem Cell Factor

Material from the C₄ column of FIG. 7 was prepared for amino acidcomposition analysis by concentration and buffer exchange into 50 mMammonium bicarbonate.

Two 70 μl samples were separately hydrolyzed in 6 N HCl containing 0.1%phenol and 0.05% 2-mercaptoethanol at 110° C. in vacuo for 24 h. Thehydrolysates were dried, reconstituted into sodium citrate buffer, andanalyzed using ion exchange chromatography (Beckman Model 6300 aminoacid analyzer). The results are shown in Table 3. Using 164 amino acids(from the protein sequencing data) to calculate amino acid compositiongives a better match to predicted values than using 193 amino acids (asdeduced from PCR-derived DNA sequencing data, FIG. 14C).

TABLE 3 Quantitative Amino Acid Composition of Mammalian Derived SCFAmino Acid Composition Predicted Moles per mole of protein¹ Residues permolecule² Amino Acid Run #1 Run #2 (A) (B) Asx 24.46 24.26 25 28 Thr10.37 10.43 11 12 Ser 14.52 14.30 16 24 Glx 11.44 11.37 10 10 Pro 10.9010.85 9 10 Gly 5.81 6.20 4 5 Ala 8.62 8.35 7/8 8 Cys nd nd 4 5 Val 14.0313.96 15 15 Met 4.05 3.99 6 7 Ile 8.31 8.33 9 10 Leu 17.02 16.97 16 19Tyr 2.86 2.84 3 7 Phe 7.96 7.92 8 8 His 2.11 2.11 2 3 Lys 10.35 11.28 1214 Trp nd nd 1 1 Arg 4.93 4.99 5 6 Total 158 158 164/165 193 Calculatedmolecular weight 18,424³ ¹Based on 158 residues from protein sequenceanalysis (excluding Cys and Trp). ²Theoretical values calculated fromprotein sequence data (A) or from DNA sequence data (B). ³Based on 1-164sequence.

Inclusion of a known amount of an internal standard in the amino acidcomposition analyses also allowed quantitation of protein in the sample;a value of 0.117 mg/ml was obtained for the sample analyzed.

EXAMPLE 3 Cloning of the Genes for Rat and Human SCF

A. Amplification and Sequencing of Rat SCF cDNA Fragments

Determination of the amino acid sequence of fragments of the rat SCFprotein made it possible to design mixed sequence oligonucleotidesspecific for rat SCF. The oligonucleotides were used as hybridizationprobes to screen rat cDNA and genomic libraries and as primers inattempts to amplify portions of the cDNA using polymerase chain reaction(PCR) strategies ([Mullis et al., Methods in Enzymol. 155, 335-350(1987)]. The oligodeoxynucleotides were synthesized by thephosphoramidite method [Beaucage, et al., Tetrahedron Lett., 22,1859-1862 (1981); McBride, et al., Tetrahedron Lett., 24, 245-248(1983)]; their sequences are depicted in FIG. 12A. The letters representA, adenine; T, thymine, C, cytosine; G, guanine; I, inosine. The * inFIG. 12A represents oligonucleotides which contain restrictionendonuclease recognition sequences. The sequences are written 5′→3′.

A rat genomic library, a rat liver cDNA library, and two BRL cDNAlibraries were screened using ³²P-labelled mixed oligonucleotide probes,219-21 and 219-22 (FIG. 12A), whose sequences were based on amino acidsequence obtained as in Example 2. No SCF clones were isolated in theseexperiments using standard methods of cDNA cloning [Maniatis, et al.,Molecular Cloning, Cold Spring Harbor 212-246 (1982)].

An alternate approach which did result in the isolation of SCF nucleicacid sequences involved the use of PCR techniques. In this methodology,the region of DNA encompassed by two DNA primers is amplifiedselectively in vitro by multiple cycles of replication catalysed by asuitable DNA polymerase (such as TaqI DNA polymerase) in the presence ofdeoxynucleotide triphosphates in a thermo cycler. The specificity of PCRamplification is based on two oligonucleotide primers which flank theDNA segment to be amplified and hybridize to opposite strands. PCR withdouble-sided specificity for a particular DNA region in a complexmixture is accomplished by use of two primers with sequencessufficiently specific to that region. PCR with single-sided specificityutilizes one region-specific primer and a second primer which can primeat target sites present on many or all of the DNA molecules in aparticular mixture [Loh et al., Science, 243, 217-220 (1989)].

The DNA products of successful PCR amplification reactions are sourcesof DNA sequence information [Gyllensten, Biotechniques, 7, 700-708(1989)] and can be used to make labeled hybridization probes possessinggreater length and higher specificity than oligonucleotides probes. PCRproducts can also be designed, with appropriate primer sequences, to becloned into plasmid vectors which allow the expression of the encodedpeptide product.

The basic strategy for obtaining the DNA sequence of the rat SCR cDNA isoutlined in FIG. 13A. The small arrows indicate PCR amplification andthe thick arrows indicate DNA sequencing reactions. PCRs 90.6 and 96.2,in conjunction with DNA sequencing, were used to obtain partial nucleicacid sequence for the rat SCR cDNA. The primers used in these PCRs weremixed oligonucleotides based on amino acid sequence depicted in FIG. 11.Using the sequence information obtained from PCRs 90.6 and 96.2, uniquesequence primer (224-27 and 224-28, FIG. 12A) were made and used insubsequent amplifications and sequencing reactions. DNA containing the5′ end of the cDNA was obtained in PCRs 90.3, 96.6, and 625.1 usingsingle-sided specificity PCR. Additional DNA sequence near theC-terminus of SCF protein was obtained in PCR 90.4. DNA sequence for theremainder of the coding region of rat SCF cDNA was obtained from PCRproducts 630.1, 630.2, 84.1 and 84.2 as described below in section C ofthis Example. The techniques used in obtaining the rat SCF cDNA aredescribed below.

RNA was prepared from BRL cells as described by Okayama et al. [MethodsEnzymol., 154, 3-28 (1987)]. PolyA+ RNA was isolated using an oligo(dT)cellulose column as described by Jacobson in [Methods in Enzymology,volume 152, 254-261 (1987)].

First-strand cDNA was synthesized using 1 μg of BRL polyA+ RNA astemplate and (dT)₁₂₋₁₈ as primer according to the protocol supplied withthe enzyme, Mo-MLV reverse transcriptase (Bethesda ResearchLaboratories). RNA strand degradation was performed using 0.14 M NaOH at84° C. for 10 min or incubation in a boiling water bath for 5 min.Excess ammonium acetate was added to neutralize the solution, and thecDNA was first extracted with phenol/chloroform, then extracted withchloroform/iso-amyl alcohol, and precipitated with ethanol. To makepossible the use of oligo(dC)-related primers in PCRs with single-sidedspecificity, a poly(dG) tail was added to the 3′ terminus of an aliquotof the first-strand cDNA with terminal transferase from calf thymus(Boeringer Mannheim) as previously described [Deng et al., MethodsEnzymol., 100, 96-103 (1983)].

Unless otherwise noted in the description which follow, the denaturationstep in each PCR cycle was set at 94° C., 1 min; and elongation was at72° C. for 3 or 4 min. The temperature and duration of annealing wasvariable from PCR to PCR, often representing a compromise based on theestimated requirements of several different PCRs being carried outsimultaneously. When primer concentrations were reduced to lessen theaccumulation of primer artifacts [Watson, Amplifications, 2, 56 (1989)],longer annealing times were indicated; when PCR product concentrationwas high, shorter annealing times and higher primer concentrations wereused to increase yield. A major factor in determining the annealingtemperature was the estimated T_(d) of primer-target association [Suggset al., in Developmental Biology Using Purified Genes eds. Brown, D. D.and Fox, C. F. (Academic, New York) pp. 683-693 (1981)]. The enzymesused in the amplifications were obtained from either of threemanufacturers: Stratagene, Promega, or Perkin-Elmer Cetus. The reactioncompounds were used as suggested by the manufacturer. The amplificationswere performed in either a Coy Tempcycle or a Perkin-Elmer Cetus DNAthermocycler.

Amplification of SCF cDNA fragments was usually assayed by agarose gelelectrophoresis in the presence of ethidium bromide and visualization byfluorescence of DNA bands stimulated by ultraviolet irradiation. In somecases where small fragments were anticipated, PCR products were analyzedby polyacrylamide gel electrophoresis. Confirmation that the observedbands represented SCF cDNA fragments was obtained by observation ofappropriate DNA bands upon subsequent amplification with one or moreinternally-nested primers. Final confirmation was by dideoxy sequencing[Sanger et al., Proc. Natl. Acad. Sci. USA, 74, 5463-5467 (1977)] of thePCR product and comparison of the predicted translation products withSCF peptide sequence information.

In the initial PCR experiments, mixed oligonucleotides based on SCFprotein sequence were used [Gould, Proc. Natl. Acad. Sci. USA, 86,1934-1938 (1989)]. Below are descriptions of the PCR amplification thatwere used to obtain DNA sequence information for the rat cDNA encodingamino acids −25 to 162.

In PCR 90.6, BRL cDNA was amplified with 4 pmol each of 222-11 and 223-6in a reaction volume of 20 μl. An aliquot of the product of PCR 90.6 waselectrophoresed on an agarose gel and a band of about the expected sizewas observed. One μl of the PCR 90.6 product was amplified further with20 pmol each of primers 222-11 and 223-6 in 50 μl for 15 cycles,annealing at 45° C. A portion of this product was then subjected to 25cycles of amplification in the presence of primers 222-11 and 219-25(PCR 96.2), yielding a single major product band upon agarose gelelectrophoresis. Asymmetric amplification of the product of PCR 96.2with the same two primers produced a template which was successfullysequenced. Further selective amplification of SCF sequences in theproduct of 96.2 was performed by PCR amplification of the product in thepresence of 222-11 and nested primer 219-21. The product of this PCR wasused as a template for asymmetric amplification and radiolabelled probeproduction (PCR2).

To isolate the 5′ end of the rat SCF cDNA, primers containing (dC)_(n)sequences, complimentary to the poly(dG) tails of the cDNA, wereutilized as non-specific primers. PCR 90.3 contained (dC)₁₂ (10 pmol)and 223-6 (4 pmol) as primers and BRL cDNA as template. The reactionproduct acted like a very high molecular weight aggregate, remainingclose to the loading well in agarose gel electrophoresis. One μl of theproduct solution was further amplified in the presence of 25 pmol of(dC)₁₂ and 10 pmol 223-6 in a volume of 25 ul for 15 cycles, annealingat 45° C. One-half μl of this product was then amplified for 25 cycleswith internally nested primer 219-25 and 201-7 (PCR 96.6). The sequenceof 201-7 is shown in FIG. 12C. No bands were observed by agarose gelelectrophoresis. Another 25 cycles of PCR, annealing at 40° C., wereperformed, after which one prominent band was observed. Southernblotting was carried out and a single prominent hybridizing band wasobserved. An additional 20 cycles of PCR (625.1), annealing at 45° C.,were performed using 201-7 and nested primer 224-27. Sequencing wasperformed after asymmetric amplification by PCR, yielding sequence whichextended past the putative amino terminus of the presumed signal peptidecoding sequence of pre-SCF. This sequence was used to designoligonucleotide primer 227-29 containing the 5′ end of the coding regionof the rat SCF cDNA. Similarly, the 3′ DNA sequence ending at amino acid162 was obtained by sequencing PCR 90.4 (see FIG. 13.A).

The sequence of the rat SCF coding region downstream of codon 162 wasobtained by direct sequencing of the products of PCRs in which rat SCF(+)− strand primers were combined with (−)− strand primers designed fromthe human SCF 3′-untranslated region sequence. Rat SCF primers 224-24(FIG. 12A) or 227-31 (5′-CTGCAGTTTGTATCTGAAG-3′) were used incombination with either of the two human SCF primers 283-19(5′-CTGCAGTTTGTATCTGAAG-3′) or 283-20 (5′-CATATAAAGTCATGGGTAG-3′). Therat SCF cDNA sequence is shown in FIG. 14C.

B. Cloning of the Rat Stem Cell Factor Genomic DNA

Probes made from PCR amplification of cDNA encoding rat SCF as describedin section A above were used to screen a library containing rat genomicsequences (obtained from CLONTECH Laboratories, Inc.; catalog numberRL1022 j). The library was constructed in the bacteriophage λ vectorEMBL-3 SP6/T7 using DNA obtained from an adult male Sprague-Dawley rat.The library, as characterized by the supplier, contains 2.3×10⁶independent clones with an average insert size of 16 kb.

PCRs were used to generate ³²P-labeled probes used in screening thegenomic library. Probe PCR1 (FIG. 13A) was prepared in a reaction whichcontained 16.7 μM ³²P[alpha]-dATP, 200 μM dCTP, 200 μM dGTP, 200 μMdTTP, reaction buffer supplied by Perkin Elmer Cetus, Taq polymerase(Perkin Elmer Cetus) at 0.05 units/ml, 0.5 μM 219-26, 0.05 μM 223-6 and1 μl of template 90.1 containing the target sites for the two primers.Probe PCR 2 was made using similar reaction conditions except that theprimers and template were changed. Probe PCR 2 was made using 0.5 μM222-11, 0.05 μM 219-21 and 1 μl of a template derived from PCR 96.2.

Approximately 10⁶ bacteriophage were plated as described in Maniatis etal. [supra (1982)]. The plaques were transferred to GeneScreen Plus™filters (22 cm×22 cm; NEN/DuPont) which were denatured, neutralized anddried as described in a protocol from the manufacturer. Two filtertransfers were performed for each plate.

The filters were prehybridized in 1 M NaCl, 1% SDS, 0.1% bovine serumalbumin, 0.1% ficoll, 0.1% polyvinylpyrrolidone (hybridization solution)for approximately 16 h at 65° C. and stored at −20° C. The filters weretransfered to fresh hybridization solution containing ³²P-labeled PCR 1probe at 1.2×10⁵ cpm/ml and hybridized for 14 h at 65° C. The filterswere washed in 0.9 M NaCl, 0.09 M sodium citrate, 0.1% SDS, pH 7.2 (washsolution) for 2 h at room temperature followed by a second wash in freshwash solution for 30 min at 65° C. Bacteriophage clones from the areasof the plates corresponding to radioactive spots on autoradiogram weeremoved from the plates and rescreened with probes PCR1 and PCR2.

DNA from positive clones was digested with restriction endonucleasesBamHI, SphI or SstI, and the resulting fragments were subcloned intopUC119 and subsequently sequenced. The strategy for sequencing the ratgenomic SCF DNA is shown schematically in FIG. 14A. In this figure, theline drawing at the top represents the region of rat genomic DNAencoding SCF. The gaps in the line indicate regions that have not beensequenced. The large boxes represent exons for coding regions of the SCFgene with the corresponding encoded amino acids indicated above eachbox. The arrows represent the individual regions that were sequenced andused to assemble the consensus sequence for the rat SCF gene. Thesequence for rat SCF gene is shown in FIG. 14B.

Using PCR 1 probe to screen the rat genomic library, clonescorresponding to exons encoding amino acids 19 to 176 of SCF wereisolated. To obtain clones for exons upstream of the coding region foramino acid 19, the library was screened using oligonucleotide probe228-30. The same set of filters used previously with probe PCR 1 wereprehybridized as before and hybridized in hybridization solutioncontaining ³²P-labeled oligonucleotide 228-30 (0.03 picomole/ml) at 50°C. for 16 h. The filters were washed in wash solution at roomtemperature for 30 min followed by a second wash in fresh wash solutionat 45° C. for 15 min. Bacteriophage clones from the areas of the platescorresponding to radioactive spots on autoradiograms were removed fromthe plates and rescreened with probe 228-30. DNA from positive cloneswas digested with restriction endonucleases and subcloned as before.Using probe 228-30, clones corresponding to the exon encoding aminoacids −20 to 18 were obtained.

Several attempts were made to isolate clones corresponding to theexon(s) containing the 5′-untranslated region and the coding region foramino acids −25 to −21. No clones for this region of the rat SCF genehave been isolated.

C. Cloning Rat cDNA for Expression in Mammalian Cells

Mammalian cell expression systems were devised to ascertain whether anactive polypeptide product of rat SCF could be expressed in and secretedby mammalian cells. Expression systems were designed to expresstruncated versions of rat SCF (SCF¹⁻¹⁶² and SCF¹⁻¹⁶⁴) and a protein(SCF¹⁻¹⁹³) predicted from the translation of the gene sequence in FIG.14C.

The expression vector used in these studies was a shuttle vectorcontaining pUC119, SV40 and HTLVI sequences. The vector was designed toallow autonomous replication in both E. coli and mammalian cells and toexpress inserted exogenous DNA under the control of viral DNA sequences.This vector, designated V19.8, harbored in E. coli DH5, is depositedwith the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md. (ATCC# 68124). This vector is a derivative of pSVDM19described in Souza U.S. Pat. No. 4,810,643 hereby incorporated byreference.

The cDNA for rat SCF⁻⁻¹⁶² was inserted into plasmid vector V19.8. thecDNA sequence is shown in FIG. 14C. The cDNA that was used in thisconstruction was synthesized in PCR reactions 630.1 and 630.2, as shownin FIG. 13A. These PCRs represent independent amplifications andutilized synthetic oligonucleotide primers 227-29 and 227-30. Thesequence for these primers was obtained from PCR generated cDNA asdescribed in section A of this Example. The reactions, 50 μl in volume,consisted of 1×reaction buffer (from a Perkin Elmer Cetus kit), 250 μMdATP, 250 μM dCTP, 250 μM dGTP, and 250 μM dTTP, 200 ng oligo(dT)-primedcDNA, 1 picomole of 227-29, 1 picomole of 227-30, and 2.5 units of Taqpolymerase (Perkin Elmer Cetus). The cDNA was amplified for 10 cyclesusing a denaturation temperature of 94° C. for 1 min, an annealingtemperature of 37° C. for 2 min, and an elongation temperature of 72° C.for 1 min. After these initial rounds of PCR amplification, 10 picomolesof 227-29 and 10 picomoles of 227-30 were added to each reaction.Amplifications were continued for 30 cycles under the same conditionswith the exception that the annealing temperature was changed to 55° C.The products of the PCR were digested with restriction endonucleasesHindIII and SstII. V19.8 was similarly digested with HindIII and SstII,and in one instance, the digested plasmid vector was treated with calfintestinal alkaline phosphatase; in other instances, the large fragmentfrom the digestion was isolated from an agarose gel. The cDNA wasligated to V19.8 using T4 polynucleotide ligase. The ligation productswere transformed into competent E. coli strain DH5 as described[Okayama, et. al., supra (1987)]. DNA prepared from individual bacterialclones was sequenced by the Sanger dideoxy method. FIG. 17 shows aconstruct of V19.8 SCF. These plasmids were used to transfect mammaliancells as described in Example 4 and Example 5.

The expression vector for rat SCF¹⁻¹⁶⁴ was constructed using a strategysimilar to that used for SCF¹⁻¹⁶² in which cDNA was synthesized usingPCR amplification and subsequently inserted into V19.8. The cDNA used inthe constructions was synthesized in PCR amplifications with V19.8containing SCF¹⁻¹⁶² cDNA (V19.8:SCF¹⁻¹⁶²) as template, 227-29 as theprimer for the 5′-end of the gene and 237-19 as the primer for the3′-end of the gene. Duplicate reactions (50 ul) contained 1×reactionbuffer, 250 uM each of dATP, dCTP, dGTP and dTTP, 2.5 units of Taqpolymerase, 20 ng of V19.8:SCF¹⁻¹⁶², and 20 picomoles of each primer.The cDNA was amplified for 35 cycles using a denaturation temperature of94° C. for 1 min, an annealing temperature of 55° C. for 2 min and anelongation temperature of 72° C. for 2 min. The products of theamplifications were digested with restriction endonucleases HindIII andSstII and inserted into V19.8. The resulting vector contains the codingregion for amino acids −25 to 164 of SCF followed by a terminationcodon.

The cDNA for a 193 amino acid form of rat SCF, (rat SCF¹⁻¹⁹³ ispredicted from the translation of the DNA sequence in FIG. 14C) was alsoinserted into plasmid vector V19.8 using a protocol similar to that usedfor the rat SCF¹⁻¹⁶². The cDNA that was used in this construction wassynthesized in PCR reactions 84.1 and 84.2 (FIG. 13A) utilizingoligonucleotides 227-29 and 230.25. The two reactions representindependent amplifications starting from different RNA preparations. Thesequence for 227-29 was obtained via PCR reactions as described insection A of this Example and the sequence for primer 230-25 wasobtained from rat genomic DNA (FIG. 14B). The reactions, 50 μl involume, consisted of 1×reaction buffer (from a Perkin Elmer Cetus kit),250 μM dATP, 250 μM dCTP, 250 μM dGTP, and 250 μM dTTP, 200 ngoligo(dT)-primed cDNA, 10 picomoles of 227-29, 10 picomoles of 230-25,and 2.5 units of Taq polymerase (Perkin Elmer Cetus). The cDNA wasamplified for 5 cycles using a denaturation temperature of 94° C. for 1½minutes, an annealing temperature of 50° C. for 2 min, and an elongationtemperature of 72° C. for 2 min. After these initial rounds, theamplifications were contained for 35 cycles under the same conditionswith the exception that the annealing temperature was changed to 60° C.The products of the PCR amplification were digested with restrictionendonucleases HindIII and SstII. V19.8 DNA was digested with HindIII andSstII and the large fragment from the digestion was isolated from anagarose gel. The cDNA was ligated to V19.8 using T4 polynucleotideligase. The ligation products were transformed into competent E. colistrain DH5 and DNA prepared from individual bacterial clones wassequenced. These plasmids were used to transfect mammalian cells inExample 4.

D. Amplification and Sequencing of Human SCF cDNA PCR Products

The human SCF cDNA was obtained from a hepatoma cell line HepG2 (ATCC HB8065) using PCR amplification as outlined in FIG. 13B. The basicstrategy was to amplify human cDNA by PCR with primers whose sequencewas obtained from the rat SCF cDNA.

RNA was prepared as described by Maniatis et al. [supra (1982)]. PolyA+RNA was prepared using oligo dT cellulose following manufacturersdirections. (Collaborative Research Inc.).

First strand cDNA was prepared as described above for BRL cDNA, exceptthat synthesis was primed with 2 μM oligonucleotide 228-28, shown inFIG. 12C, which contains a short random sequence at the 3′ end attachedto a longer unique sequence. The unique-sequence portion of 228-28provides a target site for amplification by PCR with primer 228-29 asnon-specific primer. Human cDNA sequences related to at least part ofthe rat SCF sequence were amplified from the HepG2 cDNA by PCR usingprimers 227-29 and 228-29 (PCR 22.7, see FIG. 13B; 15 cycles annealingat 60° C. followed by 15 cycles annealing at 55° C.). Agarose gelelectrophoresis revealed no distinct bands, only a smear of apparentlyheterogeneously sized DNA. Further preferential amplification ofsequences closely related to rat SCR cDNA was attempted by carrying outPCR with 1 μl of the PCR 22.7 product using internally nested rat SCFprimer 222-11 and primer 228-29 (PCR 24.3; 20 cycles annealing at 55°C.). Again only a heterogeneous smear of DNA product was observed onagarose gels. Double-sided specific amplification of the PCR 24.3products with primers 222-11 and 227-30 (PCR 25.10; 20 cycles) gave riseto a single major product band of the same size as the corresponding ratSCF cDNA PCR product. Sequencing of an asymmetric PCR product (PCR 33.1)DNA using 224-24 as sequencing primer yielded about 70 bases of humanSCF sequences.

Similarly, amplifications of 1 μl of the PCR 22.7 product, first withprimers 224-25 and 228-29 (PCR 24.7, 20 cycles), then with primers224-25 and 227-30 (PCR 41.11) generated one major band of the same sizeas the corresponding rat SCF product, and after asymmetric amplification(PCR 42.3) yielded a sequence which was highly homologous to the rat SCFsequence when 224-24 was used as sequencing primer. Unique sequenceoligodeoxynucleotides targeted at the human SCF cDNA were synthesizedand their sequences are given in FIG. 12B.

To obtain the human counterpart of the rat SCF PCR-generated codingsequence which was used in expression and activity studies, a PCR withprimers 227-29 and 227-30 was performed on 1 μl of PCR 22.7 product in areaction volume of 50 μl (PCR 39.1). Amplification was performed in aCoy Tempcycler. Because the degree of mismatching between the human SCFcDNA and the rat SCF unique primer 227-30 was unknown, a low stringencyof annealing (37° C.) was used for the first three cycles; afterwardannealing was at 55° C. A prominent band of the same size (about 590 bp)as the rat homologue appeared, and was further amplified by dilution ofa small portion of PCR 39.1 product and PCR with the same primers (PCR41.1). Because more than one band was observed in the products of PCR41.1, further PCR with nested internal primers was performed in order todetermine at least a portion of its sequence before cloning. After 23cycles of PCR with primers 231-27 and 227-29 (PCR 51.2), a single,intense band was apparent. Asymmetric PCRs with primers 227-29 and231-27 and sequencing confirmed the presence of the human SCF cDNAsequences. Cloning of the PCR 41.1 SCF DNA into the expression vectorV19.8 was performed as already described for the rat SCF 1-162 PCRfragments in Section C above. DNA from individual bacterial clones wassequenced by the Sanger dideoxy method.

E. Cloning of the Human Stem Cell Factor Genomic DNA

A PCR7 probe made from PCR amplification of cDNA, see FIG. 13B, was usedto screen a library containing human genomic sequences. A riboprobecomplementary to a portion of human SCF cDNA, see below, was used tore-screen positive plaques. PCR 7 probe was prepared starting with theproduct of PCR 41.1 (see FIG. 13B). The product of PCR 41.1 was furtheramplified with primers 227-29 and 227-30. The resulting 590 bp fragmentwas eluted from an agarose gel and reamplified with the same primers(PCR 58.1). The product of PCR 58.1 was diluted 1000-fold in a 50 μlreaction containing 10 pmoles 233-13 and amplified for 10 cycles. Afterthe addition of 10 pmoles of 227-30 to the reaction, the PCR wascontinued for 20 cycles. An additional 80 pmoles of 233-13 was added andthe reaction volume increased to 90 μl and the PCR was continued for 15cycles. The reaction products were diluted 200-fold in a 50 μl reaction,20 pmoles of 231-27 and 20 pmoles of 233-13 were added, and PCR wasperformed for 35 cycles using an annealing temperature of 43° inreaction 96.1. To produce ³²P-labeled PCR7, reaction conditions similarto those used to make PCR1 were used with the following exceptions: in areaction volume of 50 μl, PCR 96.1 was diluted 100-fold; 5 pmoles of231-27 was used as the sole primer; and 45 cycles of PCR were performedwith denaturation at 94° for 1 minute, annealing at 48° for 2 minutesand elongation at 72° for 2 minutes.

The riboprobe, riboprobe 1, was a ³²P-labelled single-stranded RNAcomplementary to nucleotides 2-436 of the hSCF DNA sequence shown inFIG. 15B. To construct the vector for the production of this probe, PCR41.1 (FIG. 13B) product DNA was digested with HindIII and EcoRI andcloned into the polylinker of the plasmid vector pGEM3 (Promega,Madison, Wis.). The recombinant pGEM3:hSCF plasmid DNA was thenlinearized by digestion with HindIII. ³²P-labeled riboprobe 1 wasprepared from the linearized plasmid DNA by runoff transcription with T7RNA polymerase according to the instructions provided by Promega. Thereaction (3 μl) contained 250 ng of linearized plasmid DNA and 20 μM³²P-rCTP (catalog #NEG-008H, New England Nuclear (NEN) with noadditional unlabeled CTP.

The human genomic library was obtained from Strategene (La Jolla,Calif.; catalog #:946203). The library was constructed in thebacteriophage Lambda Fix II vector using DNA prepared from a Caucasianmale placenta. The library, as characterized by the supplier, contained2×10⁶ primary plaques with an average insert size greater than 15 kb.Approximately 10⁶ bacteriophage were plated as described in Maniatis, etal. [supra (1982)]. The plaques were transferred to Gene Screen Plus®filters (22 cm²; NEN/DuPont) according to the protocol from themanufacturer. Two filter transfers were performed for each plate.

The filters were prehybridized in 6×SSC (0.9 M NaCl, 0.09 M sodiumcitrate pH 7.5), 1% SDS at 60° C. The filters were hybridized in fresh6×SSC, 1% SDS solution containing ³²P-labeled PCR 7 probe at 2×10⁵cpm/ml and hybridized for 20 h at 62° C. the filters were washed in6×SSC, 1% SDS for 16 h at 62° C. A bacteriophage plug was removed froman area of a plate which correspond to radioactive spots onautoradiograms and rescreened with probe PCR 7 and riboprobe 1. Therescreen with PCR 7 probe was performed using conditions similar tothose used in the initial screen. The rescreen with riboprobe 1 wasperformed as follows: the filters were prehybridized in 6×SSC, 1% SDSand hybridized at 62° C. for 18 h in 0.25 M NaPO₄, (pH 7.5), 0.25 MNaCl, 0.001 M EDTA, 15% formamide, 7% SDS and riboprobe at 1×10⁶ cpm/ml.The filters were washed in 6×SSC, 1% SDS for 30 min at 62° C. followedby 1×SSC, 1% SDS for 30 min at 62° C. DNA from positive clones wasdigested with restriction endonucleases BAM HI, SphI or SstI and theresulting fragments were subcloned into pUC119 and subsequentlysequenced.

Using probe PCR 7, a clone was obtained that included exons encodingamino acids 40 to 176 and this clone is deposited at the ATCC (deposit#40681). To obtain clones for additional SCF exons, the human genomiclibrary was screened with riboprobe 2 and oligonucleotide probe 235-29.The library was screened in a manner similar to that done previouslywith the following exceptions: the hybridization with probe 235-29 wasdone at 37° C. and the washed for this hybridization were for 1 h at 37°C. and 1 h at 44° C. Positive clones were rescreened with riboprobe 2,riboprobe 3 and oligonucleotide probes 235-29 and 236-31. Riboprobes 2and 3 were made using a protocol similar to that used to produceriboprobe 1, with the following exceptions: (a) the recombinantpGEM3:hSCF plasmid DNA was linearized with restriction endonucleasePvuII (riboprobe 2) or PstI (riboprobe 3) and (b) the SP6 RNA polymerase(Promega) was used to synthesize riboprobe 3.

FIG. 15A shows the strategy used to sequence human genomic DNA. In thisfigure, the line drawing at the top represents the region of humangenomic DNA encoding SCF. The gaps in the line indicate regions thathave not been sequenced. The large boxes represent exons for codingregions of the SCF gene with the corresponding encoded amino acidsindicated above each box. The sequence of the human SCF gene is shown inFIG. 15B. The sequence of human SCF cDNA obtained PCR techniques isshown in FIG. 15C.

The sequence of exons 7, 8 and 9, which include the coding region foramino acids 177 to 248, were obtained from a bacteriophage lambda cloneisolated as described above using PCR7 as probe.

To isolate a clone of exon 1 of the human SCF gene, a second genomiclibrary was screened. The library, purchased from Clontech (Palo Alto,Calif.; catalog #HL 1067 J), was constructed in bacteriophage lambdavector EMBL3 SP6/T7 and contained 2.5×10⁶ independent clones with anaverage insert size of 15 kb. Approximately 10⁶ clones were plated andscreened as described above using oligonucleotide probe 249-31(5′-ACTTGTGTCTTCTTCATAAGGAAAGGC-3). A SacI restriction fragment of thelambda clone was cloned into plasmid vector pGEM4 for subsequentsequence analysis. The sequence of the human SCF gene including exons 1,7, 8 and 9 is shown in FIG. 15D.

F. Sequence of the Human SCF cDNA 5′ Region

Sequencing of products from PCRs primed by two gene-specific primersreveals the sequence of the region bounded by the 3′ ends of the twoprimers. One-sided PCRs, as indicated in Example 3A, can yield thesequence of flanking regions. One-sided PCR was used to extend thesequence of the 5′-untranslated region of human SCF cDNA.

First strand cDNA was prepared from poly A+ RNA from the human bladdercarcinoma cell line 5637 (ATCC HTB 9) using oligonucleotide 228-28 (FIG.12C) as primer, as described in Example 3D. Tailing of this cDNA with dGresidues, followed by one-sided PCR amplification using primerscontaining (dC)_(n) sequences in combination with SCF-specific primers,failed to yield cDNA fragments extending upstream (5′) of the knownsequence.

A small amount of sequence information was obtained from PCRamplification of products of second strand synthesis primed byoligonucleotide 228-28. The untailed 5637 first strand cDNA describedabove (about 50 ng) and 2 pmol of 228-28 were incubated with Klenowpolymerase and 0.5 mM each of dATP, dCTP, dGTP and dTTP at 10-12° C. for30 minutes in 10 uL of 1×Nick-translation buffer [Maniatis et al.,Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory(1982)]. Amplification of the resulting cDNA by sequential one-sidedPCRs with primer 228-29 in combination with nested SCF primers (in orderof use: 235-30, 233-14, 236-31 and finally 235-29) yielded complexproduct mixtures which appeared as smears on agarose gels. Significantenrichment of SCF-related cDNA fragments was indicated by the increasingintensity of the specific product band observed when comparable volumesof the successive one-sided PCR products were amplified with two SCFprimers (227-29 and 235-29, for example, yielding a product of about 150bp). Attempts to select for a particular size range of products bypunching out portions of the agarose gel smears and reamplifying by PCRin most cases failed to yield a well-defined band which containedSCF-related sequences.

One reaction, PCR 16.17, which contained only the 235-29 primer, gaverise to a band which apparently arose from priming by 235-29 at anunknown site 5′ of the coding region in addition to the expected site,as shown by mapping with the restriction enzymes PvuII and PstI and PCRanalysis with nested primers. This product was gel-purified andreamplified with primer 235-29, and sequencing was attempted by theSanger dideoxy method using ³²P-labelled primer 228-30. The resultingsequence was the basis for the design of oligonucleotide 254-9 (FIG.12B). When this 3′ directed primer was used in subsequent PCRs incombination with 5′ directed SCF primers, bands of the expected sizewere obtained. Direct Sanger sequencing of such PCR products yieldednucleotides 180 through 204 of a human SCF cDNA sequence, FIG. 15C.

In order to obtain more sequence at the 5′ end of the hSCF cDNA, firststrand cDNA was prepared from 5637 poly A⁺ RNA (about 300 ng) using anSCF-specific primer (2 pmol of 233-14) in a 16 uL reaction containing0.2 U MMLV reverse transcriptase (purchased from BRL) and 500 uM eachdNTP. After standard phenol-chloroform and chloroform extractions andethanol precipitation (from 1 M ammonium acetate) steps, the nucleicacids were resuspended in 20 uL of water, placed in a boiling water bathfor 5 minutes, then cooled and tailed with terminal transferase in thepresence of 8 uM dATP in a CoCl₂-containing buffer [Deng and Wu, Methodsin Enzymology, 100, pp. 96-103]. The product, (dA)_(n)-tailedfirst-strand cDNA was purified by phenol-chloroform extraction andethanol precipitation and resuspended in 20 uL of 10 mM tris, pH 8.0,and 1 mM EDTA.

Enrichment and amplification of human SCF-related cDNA 5′ end fragmentsfrom about 20 ng of the (dA)_(n)-tailed 5637 cDNA was performed asfollows: an initial 26 cycles of one-sided PCR were performed in thepresence of SCF-specific primer 236-31 and a primer or primer mixturecontaining (dT)_(n) sequences at or near the 3′ end, for instance primer221-12 or a mixture of primers 220-3, 220-7, and 220-11 (FIG. 12C). Theproducts (1 μl) of these PCRs were then amplified in a second set ofPCRs containing primers 221-12 and 235-29. A major product band ofapproximately 370 bp was observed in each case upon agarose gelanalysis. A gel plug containing part of this band was punched out of thegel with the tip of a Pasteur pipette and transferred to a smallmicrofuge tube. 10 uL of water was added and the plug was melted in an84° C. heating block. A PCR containing primers 221-12 and 235-29 (8 pmoleach) in 40 uL was inoculated with 2 uL of the melted, diluted gel plug.After 15 cycles, a slightly diffuse band of approximately 370 bp wasvisible upon agarose gel analysis. Asymmetric PCRs were performed togenerate top and bottom strand sequencing templates: for each reaction,4 uL of PCR reaction product and 40 pmol of either primer 221-12 orprimer 235-29 in a total reaction volume of 100 uL were subjected to 25cycles of PCR (1 minute, 95° C.; 30 seconds, 55° C.; 40 seconds, 72°C.). Direct sequencing of the 221-12 primed PCR product mixtures (afterthe standard extractions and ethanol precipitation) with ³²P-labelledprimer 262-13 (FIG. 12B) yielded the 5′ sequence from nucleotide 1 to179 (FIG. 15C).

G. Amplification and Sequencing of Human Genomic DNA at the Site of theFirst Coding Exon of the Stem Cell Factor

Screening of a human genomic library with SCF oligonucleotide probesfailed to reveal any clones containing the known portion of the firstcoding exon. An attempt was then initiated to use a one-sided PCRtechnique to amplify and clone genomic sequences surrounding this exon.

Primer extension of heat-denatured human placental DNA (purchased fromSigma) was performed with DNA polymerase I (Klenow enzyme, largefragment; Boehringer-Mannheim) using a non-SCF primer such as 228-28 or221-11 under non-stringent (low temperature) conditions, such as 12° C.,to favor priming at a very large number of different sites. Eachreaction was then diluted five-fold into TaqI DNA polymerase buffercontaining TaqI polymerase and 100 uM of each dNTP, and elongation ofDNA strands was allowed to proceed at 72° C. for 10 minutes. The productwas then enriched for stem cell factor first exon sequences by PCR inthe presence of an SCF first exon oligonucleotide (such as 254-9) andthe appropriate non-SCF primer (228-29 or 221-11). Agarose gelelectrophoresis revealed that most of the products were short (less than300 bp). To enrich for longer species, the portion of each agarose gellane corresponding to length greater than 300 bp was cut out andelectrophoretically eluted. After ethanol precipitation and resuspensionin water, the gel purified PCR products were cloned into a derivative ofpGEM4 containing an SfiI site as a HindIII to SfiI fragment.

Colonies were screened with a ³²P-labelled SCF first exonoligonucleotide. Several positive colonies were identified and thesequences of the inserts were obtained by the Sanger method. Theresulting sequence, which extends downstream from the first exon througha consensus exon-intron boundary into the neighboring intron, is shownin FIG. 15B.

H. Amplification and Sequencing of SCF cDNA Coding Regions from Mouse,Monkey, Dog, Cat, Cow and Chicken

First strand cDNA was prepared from total RNA or poly A⁺ RNA from monkeyliver (purchased from Clontech) and from the cell lines NIH-3T3 (mouse,ATCC CRL 1658), D17 (dog, ATCC CCL 183), bovine endothelial cell line(provided by Yves DeClerck, Childrens Hospital Los Angeles, Los Angeles,Calif.), feline embryonic fibroblast cell line (Jarrett et al., J. Gen.Virology, 20:169-175 (1973)) and chicken brain RNA. The primer used infirst strand cDNA synthesis was either the nonspecific primer 228-28 oran SCF primer (227-30, 237-19, 237-20, 230-25 or 241-6).

PCR amplification with primer 227-29 and one of the primers 227-30,237-19 or 237-20 in each case except chicken yielded a fragment of theexpected size which was sequenced either directly or after cloning intoV19.8 or a pGEM vector. Additional sequences near the 5′ end of the SCFcDNAs were obtained from PCR amplifications utilizing an SCF-specificprimer in combination with either 254-9 or one of the non-specificprimers 228-29 and 221-11. Additional sequences at the 3′ end of the SCFcoding regions were obtained after PCR amplification of 228-28 primedcDNA with combinations of SCF coding region (+)-strand primers with(−)-primers based on the human SCF 3′ untranslated region as describedin Example 3A. The primers 283-19 and 283-20 (Example 3A) and primer287-9 (5′-TGTACGAAAGTAACAGTGTTG-3′) were used. In the case of chicken,amplification was accomplished with primers to 227-29 or 247-1(5′-ACTGCTCCTATTTAATCCTCTC-3′) in combination with 247-2(5′-CACTGACTCTGGAATCTTTCTCA-3′) or 287-9. The aligned amino acidsequences of human (FIG. 42), monkey, dog, mouse, rat, cat, cwo andchicken. SCF mature proteins are shown in FIG. 16.

The known SCF amino acid sequences are highly homologous throughout muchof their length. Identical consensus signal peptide sequences arepresent in the coding regions of all seven species. The amino acidexpected to be at the amino terminus of the mature protein by analogywith the rat SCF is designated by the numeral 1 in this figure. The dogand cow cDNA sequence contains an ambiguity which results in avaline/leucine ambiguity in the amino acid sequence at codon 129. Thehuman, monkey, rat and mouse amino acid sequences co-align without anyinsertions or deletions. The dog sequence has a single extra residue atposition 130 as compared to the other species. Human and monkey differat only one position, a conservative replacement of valine (human) byalanine (monkey) at position 130. The predicted SCF sequence immediatelybefore and after the putative processing site near residue 164 is highlyconserved between species.

EXAMPLE 4 Expression of Recombinant Rat SCF in COS-1 Cells

For transient expression in COS-1 cells (ATCC CRL 1650), vector V19.8(Example 3C) containing the rat SCF¹⁻¹⁶² and SCF¹⁻¹⁹³ genes wastransfected into duplicate 60 mm plates [Wigler et al., Cell, 14,725-731 (1978)]. The plasmid V19.8 SCF is shown in FIG. 17. As acontrol, the vector without insert was also transfected. Tissue culturesupernatants were harvested at various time points post-transfection andassayed for biological activity. Table 4 summarizes the HPP-CFC bioassayresults and Table 5 summarizes the MC/9 ³H-thymidine uptake data fromtypical transfection experiments. Bioassay results of supernatants fromCOS-1 cells transfected with the following plasmids are shown in Tables4 and 5: a C-terminally-truncated form of rat SCF with the C-terminus atamino acid position 162 (V19.8 rat SCF¹⁻¹⁶²), SCF¹⁻¹⁶² containing aglutamic acid at position 81 [V19.8 rat SCF¹⁻¹⁶² (Glu81)], and SCF¹⁻¹⁶²containing an alanine at position 19 [V19.8 rat SCF¹⁻¹⁶² (Ala19)]. Theamino acid substitutions were the product of PCR reactions performed inthe amplification of rat SCF¹⁻¹⁶² as indicated in Example 3. Individualclones of V19.8 rat SCF¹⁻¹⁶² were sequenced and two clones were found tohave amino acid substitutions. As can be seen in Tables 4 and 5, therecombinant rat SCF (also referred to throughout this application asrrat SCF or rrSCF), is active in the bioassays used to purify naturalmammalian SCF in Example 1.

TABLE 4 HPP-CFC Assay of COS-1 Supernatants from Cells Transfected withRat SCF DNA Volume of Colony Sample CM Assayed (μl) #/200,000 cellsV19.8 (no insert) 100 0 50 0 25 0 12 0 V19.8 rat SCF¹⁻¹⁶² 100 >50 50 >5025 >50 12 >50 6 30 3 8 V19.8 rat SCF¹⁻¹⁶² 100 26 (Glu81) 50 10 25 2 12 0V19.8 rat SCF¹⁻¹⁶² 100 41 (Ala19) 50 18 25 5 12 0 6 0 3 0

TABLE 5 MC/9³H-Thymidine Uptake Assay of COS-1 Supernatants from CellsTransfected with Rat SCF DNA Sample Volume of CM Assayed (μl) cpm v19.8(no insert) 25 1,936 12 2,252 6 2,182 3 1,682 v19.8 SCF¹⁻¹⁶² 25 11,64812 11,322 6 11,482 3 9,638 v19.8 SCF¹⁻¹⁶² (Glu81) 25 6,220 12 5,384 63,692 3 1,980 v19.8 SCF¹⁻¹⁶² (Ala19) 25 8,396 12 6,646 6 4,566 3 3,182

Recombinant rat SCF, and other factors, were tested individually in ahuman CFU-GM [Broxmeyer et al., supra (1977)] assay which measures theproliferation of normal bone marrow cells and the data are shown inTable 6. Results for COS-1 supernatants from cultures 4 days aftertransfection with V19.8 SCF¹⁻¹⁶² in combination with other factors arealso shown in Table 6. Colony numbers are the average of triplicatecultures.

The recombinant rat SCF has primarily a synergistic activity on normalhuman bone marrow in the CFU-GM assay. In the experiment in Table 6, SCFsynergized with human GM-CSF, human IL-3, and human CSF-1. In otherassays, synergy was observed with G-CSF also. There was someproliferation of human bone marrow after 14 days with rat SCF; however,the clusters were composed of <40 cells. Similar results were obtainedwith natural mammalian-derived SCF.

TABLE 6 Human CFU-GM Assay of COS-1 Supernatants from Cells Transfectedwith Rat SCF DNA Sample Colony #/100,000 cells (±SEM) Saline 0 GM-CSF  7± 1 G-CSF 24 ± 1 IL-3  5 ± 1 CSF-1 0 SCF¹⁻¹⁶² 0 GM-CSF + SCF¹⁻¹⁶² 29 ± 6G-CSF + SCF¹⁻¹⁶² 20 ± 1 IL-3 + SCF¹⁻¹⁶² 11 ± 1 CSF-1 + SCF¹⁻¹⁶²  4 + 0

EXAMPLE 5 Expression of Recombinant SCF in Chinese Hamster Ovary Cells

This example relates to a stable mammalian expression system forsecretion of SCF from CHO cells (ATCC CCL 61 selected for DHFR-).

A. Recombinant Rat SCF

The expression vector used for SCF production was V19.8 (FIG. 17). Theselectable marker used to establish stable transformants was the genefor dihydrofolate reductase in the plasmid pDSVE.1. Plasmid pDSVE.1(FIG. 18) is a derivative of pDSVE constructed by digestion of pDSVE bythe restriction enzyme SalI and ligation to an oligonucleotide fragmentconsisting of the two oligonucleotides

5′TCGAC CCGGA TCCCC 3′

3′ G GGCCT AGGGG AGCT 5′.

Vector pDSVE is described in commonly owned U.S. Ser. Nos. 025,344 and152,045 hereby incorporated by reference. The vector portion of V19.8and pDSVE.1 contain long stretches of homology including a bacterialColEl origin of replication and ampicillin resistance gene and the SV40origin of replication. This overlap may contribute to homologousrecombination during the transformation process, thereby facilitatingco-transformation.

Calcium phosphate co-precipitates of V19.8 SCF constructs and pDSVE.1were made in the presence or absence of 10 μg of carrier mouse DNA using1.0 or 0.1 μg of pDSVE.1 which had been linearized with the restrictionendonuclease PvuI and 10 μg of V19.8 SCF as described [Wigler et al.,supra (1978)]. Colonies were selected based upon expression of the DHFRgene from pDSVE.1. Colonies capable of growth in the absence of addedhypoxanthine and thymidine were picked using cloning cylinders andexpanded as independent cell lines. Cell supernatants from individualcell lines were tested in an MC/9 ³H-thymidine uptake assay. Resultsfrom a typical experiment are presented in Table 7.

TABLE 7 MC/9 ³H-Thymidine Uptake Assay of Stable CHO Cell SupernatantsFrom Cells Transfected With Rat SCF DNA Volume of ConditionedTransfected DNA Medium Assayed cpm V19.8 SCF¹⁻¹⁶² 25 33,926 12 34,973 630,657 3 14,714 1.5 7,160 None 25 694 12 1,082 6 880 3 672 1 1,354

B. Recombinant Human SCF

Expression of SCF in CHO cells was also achieved using the expressionvector pDSVRα2 which is described in commonly owned Ser. No. 501,904filed Mar. 29, 1990, hereby incorporated by reference. This vectorincludes a gene for the selection and amplification of clones based onexpression of the DHFR gene. The clone pDSRα2 SCF was generated by a twostep process. The V19.8 SCF was digested with the restriction enzymeBamHI and the SCF insert was ligated into the BamHI site of pGEM3. DNAfrom pGEM3 SCF was digested with HindIII and SalI and ligated intopDSRα2 digested with HindIII and SalI. The same process was repeated forhuman genes encoding a COOH-terminus at the amino acid positions 162,164 and 183 of the sequence shown in FIG. 15C.

Genes encoding proteins with the COOH-terminus at position 248 of thesequences shown in FIG. 42 and amino acids 1-220 of the sequence in FIG.44 were generated as follows: DNA encoding the 1-164 amino acid SCFinsert in pGEM3 was isolated by digestion with HindIII and ligated intothe HindIII site of M13mp18. The sequence preceding the ATG initiationcodon was changed by site directed mutagenesis using the oligonucleotide

5′-TCTTCTTCATGGCGGCGGCAAGCTT-3′

and a kit from Amersham (Arlington Heights, Ill.). The resulting clonewas digested with HindIII and the SCF sequences were ligated to pDSRα2digested with HindIII. This clone was designated pDSRα2-Δ12. The 3′ endof this gene was exchanged with the 3′ end of the 248 or 220 sequencesby digesting pDSRα2-Δ12 with XbaI, filling in the resulting ends withDNA polymerase I (Klenow fragment) and dATP, dCTP, dGTP and TTP togenerate a blunt end and subsequent digestion with SpeI. The 220 and 248sequences were digested with DraI, which leaves a blunt end and SpeI.The vector and inserts were then ligated together to generate pDSRα2-Δ23(248 amino acid sequence) or pDSRα2-Δ220 (220 amino acid sequence).These plasmids were used to generate cell lines by calcium phosphateprecipitation as described in Example 5A except that pDSVE.1 was notused for selection.

Established cell lines were challenged with methotrexate [Shimke, inMethods in Enzymology, 151 85-104 (1987)] at 10 nM to increaseexpression levels of the DHFR gene and the adjacent SCF gene. Expressionlevels of recombinant human SCF were assayed by radioimmune assay, as inExample 7, and/or induction of colony formation in vitro using humanperipheral blood leucocytes. This assay is performed as described inExample 9 (Table 12) except that peripheral blood is used instead ofbone marrow and the incubation is performed at 20% 0₂, 5% CO₂, and 75%N₂ in the presence of human EPO (10 U/ml). Results from typicalexperiments are shown in Table 8. The SCF²²⁰ and SCF²⁴⁸ also showedsimilar expression in these assays and as determined by Western blotanalysis. The CHO clone expressing human SCF¹⁻¹⁶⁴ has been deposited onSep. 25, 1990 with ATCC (CRL 10557) and designated Hu164SCF17.

TABLE 8 hPBL Colony Assay of Conditioned Media From Stable CHO CellLines Transfected With Human SCF DNA Media Number of Transfected DNAassayed (μl) Colonies/10⁵ pDSRα2 hSCF¹⁻¹⁶⁴ 50 53 25 45 12.5 27 6.25 13pDSRα2 hSCF¹⁻¹⁶² 10 43 5 44 2.5 31 1.25 17 0.625 21 None (CHO control)50  4

C. Secreted Product of CHO Cells Transfected with pDSRα2-Δ23.

CHO cells transfected with pDSRα2-Δ23 (248 amino acid sequence; seeExample 5B) were cultured as described in Example 11A. As previouslydescribed, the sequences shown in FIG. 42 include a putative hydrophobictransmembrane region represented by amino acids numbered 190-212, whichcould anchor a synthesized protein in the cell membrane. This is alsothe case for the encoded rat sequences of FIG. 14, yet soluble rat SCFrepresenting amino acids 1-164/165 was recovered from conditioned mediumof BRL-3A cells as described in Examples 1 and 2. This is indicative ofproteolytic processing leading to release of soluble SCF. To study suchprocessing for a case involving the human protein, the CHO cellstransfected with pDSRα2-Δ23 were cultured as described in Example 5B.Conditioned medium contained soluble human SCF, which was purifiedessentially by the methods outlined in Example 11B. By SDS-PAGE,combined with the use of glycosidases as outlined in Examples 10 and11C, it was found that the behavior of the purified material was muchlike that described for BRL-3A derived rat SCF (Example 1D) and forhuman SCF purified from conditioned medium of CHO cells transfected withpDSRα2 human SCF¹⁻¹⁶² (see Example 11C). The mobility on SDS-PAGE of themajor band remaining after treatment with neuraminidase, O-glycanase,and N-glycanase was slightly less that the mobility seen for the majorband after such treatment of the CHO cell-derived human SCF 1-162described in Example 11C. This mobility difference corresponded to lessthan 1000 in molecular weight difference and indicated that the lessmobile product was larger by a few amino acids.

The purified material from the CHO cells transfected with pDSRα2-Δ23 wassubjected to detailed structural analysis, by methods including thosegiven in Example 2. The N-terminal amino acid sequence is Glu-Gly-Ile .. . , indicating that it is the product of processing/cleavage betweenresidues indicated as numbers (−1) Thr and (+1) (Glu) in FIG. 42.

To determine the precise C-terminal processing site(s), the purifiedmaterial was subjected to AspN peptidase digestion (20-50 μg SCF in100-200 μl 0.1 M sodium phosphate, pH 7.2, for 18 h at 37° C. withAspN:SCF ratio of 1:200 by weight) followed by HPLC to isolate resultingpeptides. The elution profile shown in FIG. 16C was obtained. Collectedpeptide fractions were sequenced to identify the C-terminal peptide. Apeptide eluting at 36.8 min represents the C-terminal peptide. ThesequenceAsp-Ser-Arg-Val-Ser-Val-(X)-Lys-Pro-Phe-Phe-Met-Leu-Pro-Pro-Val-Ala-(Ala)was assigned, where (X) denotes an unassigned residue, and (Ala) denotestentative assignment due to low recovery. The indicated amino acidscorresponds to position 148-165 of the sequence shown in FIG. 42.

After treatment of the C-terminal peptide with neuraminidase andO-glycanase to remove carbohydrate, fast atom bombardment—massspectroscopy (FAB-MS) analysis indicated a molecular weight of 1815.19for the protonated monoisotopic ion (NH⁺), consistent with the sequenceAsp-Ser-Arg-Val-Ser-Val-Thr-Lys-Pro-Phe-Phe-Met-Leu-Pro-Pro-Val-Ala-Ala(calculated molecular weight of MH⁺=1815.98). A less abundant ionspecies of mass 1744.37, corresponding to the above-mentioned peptidetruncated by one Ala at the C-terminus (calculated MH⁺—1744.17), wasalso detected.

Further analyses were performed using electrospray mass spectroscopy(ES-MS). The deglycosylated C-terminal peptide fraction of the CHOcell-derived SCF and the C-terminal peptide fraction from E.coli-derived SCF¹⁻¹⁶⁵ (obtained as described in Example 2) wereanalyzed. A major signal with mass 1815 and a second signal with mass1743 were detected for the peptide of CHO cell-derived SCF. Only an 1814signal was detected for the peptide of E. coli-derived SCF.

These data indicate that soluble SCF is released from CHO cellstransfected with pDSRα2-Δ23 by proteolytic cleavage after amino acid 164or 165. This processing matches that found for BRL-3A cell derived ratSCF (Example 2).

EXAMPLE 6 Expression of Recombinant SCF in E. coli

A. Recombinant Rat SCF

This example relates to expression in E. coli of SCF polypeptides bymeans of a DNA sequence encoding [Met⁻¹] rat SCF¹⁻¹⁹³ (FIG. 14C).Although any suitable vector may be employed for protein expressionusing this DNA, the plasmid chosen was pCFM1156 (FIG. 19). This plasmidcan be readily constructed from pCFM 836 (see U.S. Pat. No. 4,710,473hereby incorporated by reference) by destroying the two endogenous NdeIrestriction sites by end-filling with T4 polymerase enzyme followed byblunt end ligation and substituting the small DNA sequence between theunique ClaI and KpnI restriction sites with the small oligonucleotideshown below.

5′ CGATTTGATTCTAGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGGTAC 3′

3′ TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC 5′

Control of protein expression in the pCFM1156 plasmid is by means of asynthetic lambda P_(L) promoter which is itself under the control of atemperature sensitive lambda CI857 repressor gene [such as is providedin E. coli strains FM5 (ATCC deposit #53911) or K12ΔHtrp]. The pCFM1156vector is constructed so as to have a DNA sequence containing anoptimized ribosome binding site and initiation codon immediately 3′ ofthe synthetic PL promoter. A unique NdeI restriction site, whichcontains the ATG initiation codon, precedes a multi-restriction sitecloning cluster followed by a lambda t-oop transcription stop sequence.

Plasmid V19.8 SCF¹⁻¹⁹³ containing the rat SCF¹⁻¹⁹³ gene cloned from PCRamplified cDNA (FIG. 14C) as described in Example 3 was digested withBglII and SstII and a 603 bp DNA fragment isolated. In order to providea Met initiation codon and restore the codons for the first three aminoacid residues (Gln, Glu, and Ile) of the rat SCF polypeptide, asynthetic oligonucleotide linker

5′ TATGCAGGA 3′

3′ ACGTCCTCTAG 5′

with NdeI and BglII sticky ends was made. The small oligonucleotide andrat SCF¹⁻¹⁹³ gene fragment were inserted by ligation into pCFM1156 atthe unique NdeI and SstII sites in the plasmid shown in FIG. 19. Theproduct of this reaction is an expression plasmid, pCFM1156 ratSCF¹⁻¹⁹³.

The pCFM1156 rat SCF¹⁻¹⁹³ plasmid was transformed into competent FM5 E.coli host cells. Selection for plasmid-containing cells was on the basisof the antibiotic (kanamycin) resistance marker gene carried on thepCFM1156 vector. Plasmid DNA was isolated from cultured cells and theDNA sequence of the synthetic oligonucleotide and its junction to therat SCF gene confirmed by DNA sequencing.

To construct the plasmid pCFM1156 rat SCF¹⁻¹⁶² encoding the [Met⁻¹] ratSCF¹⁻¹⁶² polypeptide, an EcoRI to SstII restriction fragment wasisolated from V19.8 rat SCF¹⁻¹⁶² and inserted by ligation into theplasmid pCFM rat SCF¹⁻¹⁹³ at the unique EcoRI and SstII restrictionsites thereby replacing the coding region for the carboxyl terminus ofthe rat SCF gene.

To construct the plasmids pCFM1156 rat SCF¹⁻¹⁶⁴ and pCFM1156 ratSCF¹⁻¹⁶⁵ encoding the [Met⁻¹] rat SCF¹⁻¹⁶⁴ and [Met⁻¹] rat SCF¹⁻¹⁶⁵polypetides, respectively, EcoRI to SstII restriction fragments wereisolated from PCR amplified DNA encoding the 3′ end of the SCF gene anddesigned to introduce site directed changes in the DNA in the regionencoding the carboxyl terminus of the SCF gene. The DNA amplificationswere performed using the oligonucleotide primers 227-29 and 237-19 inthe construction of pCFM1156 rat SCF¹⁻¹⁶⁴ and 227-29 and 237-20 in theconstruction of pCFM1156 rat SCF¹⁻¹⁶⁵.

B. Recombinant Human SCF

This example relates to the expression in E. coli of human SCFpolypeptide by means of a DNA sequence encoding [Met⁻¹] human SCF¹⁻¹⁶⁴and [Met⁻¹] human SCF¹⁻¹⁸³ (FIG. 15C); and [Met⁻¹] human SCF¹⁻¹⁶⁵ (FIG.15C). Plasmid V19.8 human SCF¹⁻¹⁶² containing the human SCF¹⁻¹⁶² genewas used as template for PCR amplification of the human SCF gene.Oligonucleotide primers 227-29 and 237-19 were used to generate the PCRDNA which was then digested with PstI and SstII restrictionendonucleases. In order to provide a Met initiation codon and restorethe codons for the first four amino acid residues (Glu, Gly, Ile, Cys)of the human SCF polypeptide, a synthetic oligonucleotide linker

5′ TATGGAAGGTATCTGCA 3′

3′ ACCTTCCATAG 5′

with NdeI and PstI sticky ends was made. The small oligo linker and thePCR derived human SCF gene fragment were inserted by ligation into theexpression plasmid pCFM1156 (as described previously) at the unique NdeIand SstII sites in the plasmid shown in FIG. 19.

The pCFM1156 human SCF¹⁻¹⁶⁴ plasmid was transformed into competent FM5E. coli host cells. Selection for plasmid containing cells was on thebasis of the antibiotic (kanamycin) resistance marker gene carried onthe pCFM1156 vector. Plasmid DNA was isolated from cultured cells andthe DNA sequence of the human SCF gene confirmed by DNA sequencing.

To construct the plasmid pCFM1156 human SCF¹⁻¹⁸³ encoding the [Met⁻¹]human SCF¹⁻¹⁸³ (FIG. 15C) polypeptide, a EcoRI to HindIII restrictionfragment encoding the carboxyl terminus of the human SCF gene wasisolated from pGEM human SCF¹¹⁴⁻¹⁸³ (described below), a SstI to EcoRIrestriction fragment encoding the amino terminus of the human SCF genewas isolated from pCFM1156 human SCF¹⁻¹⁶⁴, and the larger HindIII toSstI restriction fragment from pCFM1156 was isolated. The three DNAfragments were ligated together to form the PCFM1156 human SCF¹⁻¹⁸³plasmid which was then tranformed into FM5 E. coli host cells. Aftercolony selection using kanamycin drug resistance, the plasmid DNA wasisolated and the correct DNA sequence confirmed by DNA sequencing. ThepGEM human SCF¹¹⁴⁻¹⁸³ plasmid is a derivative of pGEM3 that contains anEcoRI-SphI fragment that includes nucleotides 609 to 820 of the humanSCF cDNA sequence shown in FIG. 15C. The EcoRI-SphI insert in thisplasmid was isolated from a PCR that used oligonucleotide primers 235-31and 241-6 (FIG. 12B) and PCR 22.7 (FIG. 13B) as template. The sequenceof primer 241-6 was based on the human genomic sequence to the 3′ sideof the exon containing the codon for amino acid 176.

A plasmid encoding human [Met⁻¹] SCF¹⁻¹⁶⁵ was constructed as follows.Sixteen oligonucleotides were “stitched together” to create a 221 basepair fragment with EcoRl and BamHl sticky ends (FIG. 16D). Thisnucleotide sequence codes for the C-terminal 68 amino acids of humanSCF¹⁻¹⁸³ (amino acid numbering and designation as in FIG. 15C). Thecodons in this nucleotide sequence reflected those most commonly used byE. coli (i.e., optimized for expression in E. coli). In addition, aunique BstEII site is present in the fragment. The EcoRl to BamHlfragment of the human SCF¹⁻¹⁸³ DNA (FIG. 15C) was removed and replacedby the fragment containing the optimized codons. This construct wasdigested with BstEII and BamHl and the 39 base pair fragment shown inFIG. 16E was introduced. The resulting plasmid codes for human [Met⁻¹]SCF¹⁻¹⁶⁵ with the codons for the C-terminal 50 amino acis optimized forexpression in E. coli.

Another plasmid encoding human [Met⁻¹] SCF¹⁻¹⁶⁵, with the codons of FIG.15C, was also constructed, by PCR utilizing pCFM1156 human SCF¹⁻¹⁶⁴. A5′ oligonucleotide was made 5′ of the EcoRl site and a 3′oligonucleotide was made which included the final codons of the 1-164sequence plus an extra codon for the position 165 and nucleotidesthrough the SstII site. After the PCR reaction, the fragment was cutwith EcoRl and SstII, gel purified, and cloned into pCFM1156 humanSCF¹⁻¹⁶⁴ cut with EcoRl and SstII.

The generation of other expression plasmids including those encodinghuman [Met⁻¹] SCF¹⁻²⁴⁸ (sequence of FIG. 42) and encoding human [Met⁻¹]SCF¹⁻²²⁰ (sequence of FIG. 44) is described in Example 28.

C. Fermentation of E. coli producing Human SCF¹⁻¹⁶⁴ and E. coliproducing Human SCF¹⁻¹⁶⁵

Fermentations for the production of SCF¹⁻¹⁶⁴ were carried out in 16liter fermentors using an FM5 E. coli K12 host containing the plasmidpCFM 1156 human SCF¹⁻¹⁶⁴. Seed stocks of the producing culture weremaintained at −80° C. in 17% glycerol in Luria broth. For inoculumproduction, 100 μl of the thawed seed stock was transferred to 500 ml ofLuria broth in a 2 L erlenmeyer flask and grown overnight at 30° C. on arotary shaker (250 RPM).

For the production of E. coli cell paste used as starting material forthe purification of human SCF¹⁻¹⁶⁴ outlines in Example 10, the followingfermentation conditions were used.

The inoculum culture was aseptically transferred to a 16 L fermentorcontaining 8 L of batch medium (see Table 9). The culture was grown inbatch mode until the OD-600 of the culture was approximately 3-5. Atthis time, a sterile feed (Feed 1, Table 10) was introduced into thefermentor using a peristaltic pump to control the feed rate. The feedrate was increased exponentially with time to give a growth rate of 0.15hr⁻¹. The temperature was controlled at 30° C. during the growth phase.The dissolved oxygen concentration in the fermentor was automaticallycontrolled at 50% saturation using air flow rate, agitation rate, vesselback pressure and oxygen supplementation for control. The pH of thefermentor was automatically controlled at 7.0 using phosphoric acid andammonium hydroxide. At an OD-600 of approximately 30, the productionphase of the fermentation was induced by increasing the fermentortemperature to 42° C. At the same time the addition of Feed 1 wasstopped and the addition of Feed 2 (Table 11) was started at a rate of200 ml/hr. Approximately six hours after the temperature of thefermentor was increased, the fermentor contents were chilled to 15° C.The yield of SCF¹⁻¹⁶⁴ was approximately 30 mg/OD-L. The cell pellet wasthen harvested by centrifugation in Beckman J6-B rotor at 3000×g for onehour. The harvested cell paste was stored frozen at −70° C.

An advantageous method for production of SCF¹⁻¹⁶⁴ is similar to themethod described above except for the following modifications.

1) The addition of Feed 1 is not initiated until the OD-600 of theculture reaches 5-6.

2) The rate of addition of Feed 1 is increased more slowly, resulting ina slower growth rate (approximately 0.08).

3) The culture is induced at OD-600 of 20.

4) Feed 2 is introduced into the fermentor at a rate of 300 mL/hr.

All other operations are similar to the method described above,including the media.

Using this process, yields of SCF¹⁻¹⁶⁴ approximately 35-40 mg/OD-L atOD=25 have been obtained.

TABLE 9 Composition of Batch Medium Yeast extract 10^(a) g/L Glucose 5K₂HPO₄ 3.5 KH₂PO₄ 4 M_(G)SO₄ · 7H₂O 1 NaCl 0.625 Dow P-2000 antifoam 5mL/8 L Vitamin solution^(b) 2 mL/L Trace metals solution^(c) 2 mL/L^(a)Unless otherwise noted, all ingredients are listed as g/L. ^(b)TraceMetals solution: FeCl₃ · 6H₂O, 27 g/L; ZnCl₂ · 4H₂O, 2g/L; CaCl₂ · 6H₂O,2 g/L; Na₂MoO₄ · 2 H₂O, 2 g/L, CuSO₄ · 5 H₂O, 1.9 g/L; concentrated HCl,100 ml/L. ^(c)Vitamin solution: riboflavin, 0.42 g/l; pantothenic acid,5.4 g/L; niacin, 6 g/L; pyridoxine, 1.4 g/L; biotin, 0.06 g/L; folicacid, 0.04 g/L.

TABLE 10 Composition of Feed Medium Yeast extract  50^(a) Glucose 450MgSO₄ · 7H₂O  8.6 Trace metals solution^(b)  10 mL/L Vitaminsolution^(c)  10 mL/L ^(a)Unless otherwise noted, all ingredients arelisted as g/L. ^(b)Trace Metals solution: FeCl₃ · 6H₂O, 27 g/L; ZnCl₂ ·4H₂O, 2g/L; CaCl₂ · 6H₂O, 2 g/L; Na₂MoO₄ · 2 H₂O, 2 g/L, CuSO₄ · 5 H₂O,1.9 g/L; concentrated HCl, 100 ml/L. ^(c)Vitamin solution: riboflavin,0.42 g/l; pantothenic acid, 5.4 g/L; niacin, 6 g/L; pyridoxine, 1.4 g/L;biotin, 0.06 g/L; folic acid, 0.04 g/L.

TABLE 11 Composition of Feed Medium 2 Tryptone 172^(a) Yeast extract  86Glucose 258 ^(a)All ingredients are listed as g/L.

For the production of E. coli cell paste used as starting material forthe purification of human SCF¹⁻¹⁶⁵ (Example 10), fermentation conditionsdiffered in the following ways from those described for the SCF¹⁻¹⁶⁴cases. Feed 1 was introduced when the OD-600 of the culture wasapproximately 5-6. Feed 1 contained 13 g/L K₂HPO₄ in addition to thecomponents listed in Table 10. The feed rate was increased exponentiallywith time to give a growth rate of 0.2 hr⁻¹. Production phase wasinduced by temperature increase at OD-600 of about 40, and the rate ofaddition of Feed 2 was 600 ml/hr. Feed 2 contained 258 g/L tryptone, 129g/L yeast extract, 50 g/L glucose, and 6.4 g/L K₂HPO₄. Chilling of thefermentor and harvesting of cells was done about eight hours after thetemperature increase.

EXAMPLE 7 Immunoassays for Detection of SCF

Radioimmunoassay (RIA) procedures applied for quantitative detection ofSCF in samples were conducted according to the following procedures.

An SCF preparation from BRL 3A cells purified as in Example 1 wasincubated together with antiserum for two hours at 37° C. After the twohour incubation, the sample tubes were then cooled on ice, ¹²⁵I-SCF wasadded, and the tubes were incubated at 4° C. for at least 20 h. Eachassay tube contained 500 μl of incubation mixture consisting of 50 μl ofdiluted antisera, ˜60,000 5 μl trasylol and 0-400 μl of SCF standard,with buffer (phosphate buffered saline, 0.1% bovine serum albumin, 0.05%Triton X-100, 0.025% azide) making up the remaining volume. Theantiserum was the second test bleed of a rabbit immunized with a 50%pure preparation of natural SCF from BRL 3A conditioned medium. Thefinal antiserum dilution in the assay was 1:2000.

The antibody-bound ¹²⁵I-SCF was precipitated by the addition of 150 μlStaph A (Calbiochem). After a 1 h incubation at room temperature, thesamples were centrifuged and the pellets were washed twice with 0.75 ml10 mM Tris-HCL pH 8.2, containing 0.15M NaCl, 2 mM EDTA, and 0.05%Triton X-100. The washed pellets were counted in a gamma counter todetermine the percent of ¹²⁵I-SCF bound. Counts bound by tubes lackingserum were subtracted from all final values to correct for nonspecificprecipitation. A typical RIA is shown in FIG. 20. The percent inhibitionof ¹²⁵I-SCF binding produced by the unlabeled standard is dose dependent(FIG. 20A), and, as indicated in FIG. 20B, when the immune precipitatedpellets are examined by SDS-PAGE and autoradiography, the ¹²⁵I-SCFprotein band is competed. In FIG. 20B, lane 1 is ¹²⁵I-SCF, and lanes 2,3, 4 and 5 are immune-precipicated ¹²⁵I-SCF competed with 0, 2, 100, and200 ng of SCF standard, respectively. As determined by both the decreasein antibody-precipitable cpm observed in the RIA tubes and decrease inthe immune-precipitated ¹²⁵I-SCF protein band (migrating atapproximately M_(r) 31,000) the polyclonal antisera recognizes the SCFstandard which was purified as in Example 1.

Western procedures were also applied to detect recombinant SCF expressedin E. coli, COS-1, and CHO cells. Partially purified E. coli expressedrat SCF¹⁻¹⁹³ (Example 10), COS-1 cell expressed rat SCF¹⁻¹⁶² andSCF¹⁻¹⁹³ as well as human SCF¹⁻¹⁶² (Examples 4 and 9), and CHO cellexpressed rat SCF¹⁻¹⁶² (Example 5), were subjected to SDS-PAGE.Following electrophoresis, the protein bands were transferred to 0.2 μmnitrocellulose using a Bio-Rad Transblot apparatus at 60V for 5 h. Thenitrocellulose filters were blocked for 4 h in PBS, pH 7.6, containing10% goat serum followed by a 14 h room temperature incubation with a1:200 dilution of either rabbit preimmune or immune serum (immunizationdescribed above). The antibody-antiserum complexes were visualized usinghorseradish peroxidase-conjugated goat anti-rabbit IgG reagents (Vectorlaboratories) and 4-chloro-1-naphtol color development reagent.

Examples of two Western analyses are presented in FIGS. 21 and 22. InFIG. 21, lanes 3 and 5 are 200 μl of COS-1 cell produced human SCF¹⁻¹⁶²;lanes 1 and 7 are 200 μl of COS-1 cell produced human EPO (COS-1 cellstransfected with V19.8 EPO); and lane 8 is prestained molecular weightmarkers. Lanes 1-4 were incubated with pre-immune serum and lanes 5-8were incubated with immune serum. The immune serum specificallyrecognizes a diffuse band with an apparent M_(r) of 30,000 daltons fromCOS-1 cells producing human SCF¹⁻¹⁶² but not from COS-1 cells producinghuman EPO.

In the Western shown in FIG. 22, lanes 1 and 7 are 1 μg of a partiallypurified preparation of rat SCF¹⁻¹⁹³ produced in E. coli, lanes 2 and 8are wheat germ agglutinin-agarose purified COS-1 cell produced ratSCF¹⁻¹⁹³; lanes 4 and 9 are wheat germ agglutinin-agarose purified COS-1cell produced rat SCF¹⁻¹⁶²; lanes 5 and 10 are wheat germagglutinin-agarose purified CHO cell produced rat SCF¹⁻¹⁶²; and lane 6is prestained molecular weight markers. Lanes 1-5 and lanes 6-10 wereincubated with rabbit preimmune and immune serum, respectively. The E.coli produced rat SCF¹⁻¹⁹³ (lanes 1 and 7) migrates with an apparentM_(r) of ˜24,000 daltons while the COS-1 cell produced rat SCF¹⁻¹⁹³(lanes 2 and 8) migrates with an apparent M_(r) of 24-36,000 daltons.This difference in molecular weights is expected since mammalian cells,but not bacteria, are capable of glycosylation. Transfection of thesequence encoding rat SCF¹⁻¹⁶² into COS-1 (lanes 4 and 9), or CHO cells(lanes 5 and 10), results in expression of SCF with a lower averagemolecular weight than that produced by transfection with SCF¹⁻¹⁹³ (lanes2 and 8).

The expression products of rat SCF¹⁻¹⁶² from COS-1 and CHO cells are aseries of bands ranging in apparent M_(r) between 24-36,000 daltons. Theheterogeneity of the expressed SCF is likely due to carbohydratevariants, where the SCF polypetide is glycosylated to different extends.

In summary, Western analyses indicate that immune serum from rabbitsimmunized with natural mammalian SCF recognize recombinant SCF producedin E. coli, COS-1 and CHO cells but fail to recognize any bands in acontrol sample consisting of COS-1 cell produced EPO. In further supportof the specificity of the SCF antiserum, preimmune serum from the samerabbit failed to react with any of the rat or human SCF expressionproducts.

Radioimmunoassay (RIA) procedures were also developed to quantify SCF inhuman serum samples. Purified CHO-derived human SCF (expression of the1-248 transcript) was used as the standard in this assay over the rangeof 0.01-10.0 ng/tube. Pooled normal human serum samples, obtained fromIrvine Scientific (Lots 500080713 and 500081015), were each assayed at25, 50, 100 and 200 μl per tube. Each tube was adjusted to contain 5 μlof trasylol, and 900 μl total volume by the addition of the appropriateamount of assay diluent (phosphate-buffered saline containing 0.1%bovine serum albumin and 0.025% sodium azide). Rabbit anti-human SCFantiserum (100 μl of a 1:50,000 dilution) was added, the tubes weremixed and incubated at 4° C. for approximately 24 hours. The antiserumwas the bleed-out of a rabbit hyperimmunized with a purified preparationof CHO-derived human SCF¹⁻¹⁶².

Following the 24 hours incubation, approximately 60,000 cpm of¹²⁵I-CHO-derived human SCF (expression of the 1-248 transcript, 57.9mCi/mg) was added to all tubes; the tubes were vortexed and incubated at4° C. for an additional 19 hours. The antibody-bound ¹²⁵I-human SCF wasprecipitated by the addition of 100 μl of a 1:50 dilution of normalrabbit serum (research Products International) and 100 μl of a 1:20dilution of goat anti-rabbit IgG (Research Products International) toall tubes. After a two hour incubation at room temperature, the tubeswere centrifuged and the pellets were washed once with 0.75 ml of 10 mMTris-HCl, pH 8.2, containing 0.15 M NaCl, 2 mM EDTA, and 0.05% TritonX-100. The washed pellets were counted in a gamma counter to determinethe percent of ¹²⁵I-human SCF bound. Counts bound by tubes lackingantiserum were subtracted from all final values to correct fornonspecific precipitation. A typical RIA is shown in FIG. 22A. Thepercent inhibition of ¹²⁵I-human SCF binding by the unlabeled standardand normal human serum was dose-dependent. Increasing aliquots of thenormal human serum, over the range of 25-200 μl produced a dose responseline which was parallel to that of the standard. Both of the normalhuman serum samples were assayed twice in this assay. Values plotted inFIG. 22A are the average percent inhibitions obtained for the respectivealiquots for each serum sample. Values of 2.16 ng/ml and 2.93 ng/ml wereobtained for SCF levels in Lot 500080713 and Lot 500081015 normal humanserum, respectively.

EXAMPLE 8 In Vivo Activity of Recombinant SCF

A. Rat SCF in Bone Marrow Transplanation

COS-1 cells were transfected with V19.8 SCF¹⁻¹⁶² in a large scaleexperiment (T175 cm² flasks instead of 60 mm dishes) as described inExample 4. Approximately 270 ml of supernatant was harvested. Thissupernatant was chromatographed on wheat germ agglutinin-agarose andS-Sepharose essentially as described in Example 1. The recombinant SCFwas evaluated in a bone marrow transplantation model based on murineW/W^(v) genetics. The W/W^(v) mouse has a stem cell defect which amongother features results in a macrocytic anemia (large red cells) andallows for the transplantation of bone marrow from normal animalswithout the need for irradiation of the recipient animals [Russel, etal., Science, 144, 844-846 (1964)]. The normal donor stem cells outgrowthe defective recipient cells after transplantation.

In the following example, each group contained six age matched mice.Bone marrow was harvested from normal donor mice and transplanted intoW/W^(v) mice. The blood profile of the recipient animals is followed atdifferent times post transplantation and engraftment of the donor marrowis determined by the shift of the peripheral blood cells from recipientto donor phenotype. The conversion from recipient to donor phenotype isdetected by monitoring the forward scatter profile (FASCAN, BectonDickenson) of the red blood cells. The profile for each transplantedanimal was compared to that for both donor and recipient untransplantedcontrol animals at each time point. The comparison was made utilizing acomputer program based on Kolmogorov-Smirnov statistics for the analysisof histograms from flow systems [Young, J. Histochem. and Cytochem., 25,935-941 (1977)]. An independent qualitative indicator of engraftment isthe hemoglobin type detected by hemoglobin electrophoresis of therecipient blood [Wong, et al., Mol. and Cell. Biol., 9, 798-808 (1989)]and agrees well with the goodness of fit determination fromKolmogorov-Smirnov statistics.

Approximately 3×10⁵ cells were transplanted without SCF treatment(control group in FIG. 23) from C56BL/6J donors into W/W^(v) recipients.A second group received 3×10⁵ donor cells which had been treated withSCF (600 U/ml) at 37° C. for 20 min and injected together (pre-treatedgroup in FIG. 23). (One unit of SCF is defined as the amount whichresults in half-maximal stimulation in the MC/9 bioassay). In a thirdgroup, the recipient mice were injected sub-cutaneously (sub-Q) withapproximately 400 U SCF/day for 3 days after transplantation of 3×10⁵donor cells (Sub-Q inject group in FIG. 23). As indicated in FIG. 23, inboth SCF-treated groups the donor marrow is engrafted faster than in theuntreated control group. By 29 days post-transplantation, the SCFpre-treated group had converted to donor phenotype. This Exampleillustrates the usefulness of SCF therapy in bone marrowtransplantation.

B. In vivo activity of Rat SCF in Steel Mice

Mutations at the S1 locus cause deficiencies in hematopoietic cells,pigment cells, and germ cells. The hematopoietic defect is manifest asreduced numbers of red blood cells [Russell, In:Al Gordon, Regulation ofHematopoiesis, Vol. I, 649-675 Appleton-Century-Crafts, New York(1970)], neutrophils [Ruscetti, Proc. Soc. Exp. Biol. Med., 152, 398(1976)], monocytes [Shibata, J. Immunol. 135, 3905 (1985)],megakaryocytes [Ebbe, Exp. Hematol., 6, 201 (1978)], natural killercells [(Clark, Immunogenetics, 12, 601 (1981)], and mast cells [Hayashi,Dev. Biol., 109, 234 (1985)]. Steel mice are poor recipients of a bonemarrow transplant due to a reduced ability to support stem cells[Bannerman, Prog. Hematol., 8, 131 (1973)]. The gene encoding SCF isdeleted in Steel (S1/S1) mice.

Steel mice provide a sensitive in vivo model for SCF activity. Differentrecombinant SCF proteins were tested in Steel-Dickie (S1/S1^(d)) micefor varying lengths of time. Six to ten week old Steel mice(WCB6Fl-S1/S1^(d)) were purchased from Jackson Labs, Bar Harbor, Me.Peripheral blood was monitored by a SYSMEX F-800 microcell counter(Baxter, Irvine, Calif.) for red cells, hemoglobin, and platelets. Forenumeration of peripheral white blood cell (WBC) numbers, a CoulterChannelyzer 256 (Coulter Electronics, Marietta, Ga.) was used.

In the experiment in FIG. 24, Steel-Dickie mice were treated with E.coli derived SCF¹⁻¹⁶⁴, purified as in Example 10, at a dose of 100μg/kg/day for 30 days, then at a dose of 30 μg/kg/day for an additional20 days. The protein was formulated in injectable saline (Abbott Labs,North Chicago, Ill.) +0.1% fetal bovine serum. The injections wereperformed daily, subcutaneously. The peripheral blood was monitored viatail bleeds of ˜50 μl at the indicated times in FIG. 24. The blood wascollected into 3% EDTA coated syringes and dispensed into powdered EDTAmicrofuge tubes (Brinkmann, Westbury, N.Y.). There is a significantcorrection of the macrocytic anemia in the treated animals relative tothe control animals. Upon cessation of treatment, the treated animalsreturn to the initial state of macrocytic anemia.

In the experiment shown in FIGS. 25 and 26, Steel-Dickie mice weretreated with different recombinant forms of SCF as described above, butat a dose of 100 μg/kg/day for 20 days. Two forms of E. coli derived ratSCF, SCF¹⁻¹⁶⁴ and SCF¹⁻¹⁹³, were produced as described in Example 10. Inaddition, E. coli SCF¹⁻¹⁶⁴, modified by the addition of polyethyleneglycol (SCF¹⁻¹⁶⁴ PEG25) as in Example 12, was also tested. CHO derivedSCF¹⁻¹⁶² produced as in Example 5 and purified as in Example 11, wasalso tested. The animals were bled by cardiac puncture with 3% EDTAcoated syringes and dispensed into EDTA powered tubes. The peripheralblood profiles after 20 days of treatment are shown in FIG. 25 for whiteblood cells (WBC) and FIG. 26 for platelets. The WBC differentials forthe SCF¹⁻¹⁶⁴ PEG25 group are shown in FIG. 27. There are absoluteincreases in neutrophils, monocytes, lymphocytes, and platelets. Themost dramatic effect is seen with SCF¹⁻¹⁶⁴ PEG 25.

An independent measurement of lymphocyte subsets was also performed andthe data is shown in FIG. 28. The murine equivalent of human CD4, ormarker of T helper cells, is L3T4 [Dialynas, J. Immunol., 131, 2445(1983)]. LyT-2 is a murine antigen on cytotoxic T cells [Ledbetter, J.Exp. Med., 153, 1503 (1981)]. Monoclonal antibodies against theseantigens were used to evaluate T cell subsets in the treated animals.

Whole blood was stained for T lymphocyte subsets as follows. Two hundredmicroliters of whole blood was drawn from individual animals into EDTAtreated tubes. Each sample of blood was lysed with sterile deionizedwater for 60 seconds and then made isotonic with 10× Dulbecco'sPhosphate Buffered Saline (PBS) (Gibco, Grand Island, N.Y.). This lysedblood was washed 2 times with 1× PBS (Gibco, Grand Island, N.Y.)supplemented with 0.1% Fetal Bovine Serum (Flow Laboratory, McLean, Va.)and 0.1% sodium azide. Each sample of blood was deposited into roundbottom 96 well cluster dishes and centrifuges. The cell pellet(containing 2-10×10⁵ cells) was resuspended with 20 microliters of Ratanti-Mouse L3T4 conjugated with phycoerythrin (PE) (Becton Dickinson,Mountain View, Calif.) and 20 microliters of Rat anti-Mouse Lyt-2conjugated with Fluorescein Isothiocyanate incubated on ice (4° C.) for30 minutes (Becton Dickinson). Following incubation the cells werewashed 2 times in 1× PBS supplemented as indicated aboved. Each sampleof blood was then analyzed on a FACScan cell analysis system (BectonDickinson, Mountain View, Calif.). This system was standardized usingstandard autocompensation procedures and Calibrite Beads (BectonDickinson, Mountain View, Calif.). These data indicated an absoluteincrease in both helper T cell populations as well as cytotoxic T cellnumbers.

C. In Vivo Activity of SCF in Primates

Human SCF¹⁻¹⁶⁴ expressed in E. coli (Example 6B) and purified tohomogeneity as in Example 10, was tested for in vivo biological activityin normal primates. Adult male baboons (Papio sp.) were studied in threegroups: untreated, n=3; SCF 100 ug/kg/day, n=6; and SCF 30 ug/kg/day,n=6. The treated animals received single daily subcutaneous injectionsof SCF. Blood specimens were obtained from the animals under ketaminerestraint. Specimens for complete blood count, reticulocyte count, andplatelet count were obtained on days 1, 6, 11, 15, 20 and 25 oftreatment.

All animals survived the protocol and had no adverse reactions to SCFtherapy. The white blood cell count increased in the 100 ug/kg treatedanimals as depicted in FIG. 29. The differential count, obtainedmanually from Wright Giemsa stained peripheral blood smears, is alsoindicated in FIG. 29. There was an absolute increase in neutrophils,lymphocytes, and monocytes. As indicated in FIG. 30 there was also anincrease at the 100 ug/kg dose in the hemtocrits as well as platelets.

Human SCF (hSCF¹⁻¹⁶⁴ modified by the addition of polyethylene glycol asin Example 12) was also tested in normal baboons, at a dose of 200μg/kg-day, administered by continuous intravenous infusion and comparedto the unmodified protein. The animals started SCF at day 0 and weretreated for 28 days. The results for the peripheral WBC are given in thefollowing table. The PEG modified SCF elicited an earlier rise inperipheral WBC than the unmodified SCF. The same results are obtainedwith human SCF¹⁻¹⁶⁵ modified by the addition of polyethylene glycol.

Treatment with 200 μg/kg-day hSCF¹⁻¹⁶⁴:

Animal # M88320 Animal # M88129 DAY WBC DAY WBC 0 5800 0 6800 +7 10700+7 7400 +14 12600 +14 20900 +16 22000 +21 18400 +22 31100 +23 24900 +2428100 +29 13000 +29 9600 +30 23000 +36 6600 +37 12100 +43 5600 +44 10700+51 7800

Treatment with 200 μg/kg-day PEG-hSCF¹⁻¹⁶⁴:

Animal # M88350 Animal # M89116 DAY WBC DAY WBC  −7 12400  −5  7900  −211600    0  7400  +4 24700  +6 16400  +7 20400  +9 17100 +11 24700 +1318700 +14 32600 +16 19400 +18 33600 +20 27800 +21 26400 −23 20700 +2516600 +27 20200 +28 26900 +29 18600 +32  9200 +33  7600

Human SCF¹⁻¹⁶⁵ expressed in E. coli (Example 6) and purified tohomogeneity as in Example 10B, demonstrates the same in vivo biologicalactivity in primotes as E. coli derived recombinant human SCF¹⁻¹⁶⁴.

EXAMPLE 9 In Vitro Activity of Recombinant Human SCF

A. Human bone marrow assay, murine HPP-CFC assay, and murine MC/9 assay.

The cDNA of human SCF corresponding to amino acids 1-162 obtained by PCRreactions outlines in Example 3D, was expressed in COS-1 cells asdescribed for the rat SCF in Example 4. COS-1 supernatants were assayedon human bone marrow as well as in the murine HPP-CFC and MC/9 assays.The human protein was not active at the concentrations tested in eithermurine assay; however, it was active on human bone marrow. The cultureconditions of the assay were as follows: human bone marrow from healthyvolunteers was centrifuged over Ficoll-Hypaque gradients (Pharmacia) andcultured in 2.1% methyl cellulose, 30% fetal calf serum, 6×10⁻⁵M2-mercaptoethanol, 2 mM glutamine, ISCOVE's medium (GIBCO), 20 U/mlEPO, and 1×10⁵ cells/ml for 14 days in a humidified atmospherecontaining 7% O₂, 10% CO₂, and 83% N₂. The colony numbers generated withrecombinant human and rat SCF COS-1 supernatants are indicated in Table12. Only those colonies of 0.2 mm in size or larger are indicated.

TABLE 12 Growth of Human Bone Marrow Colonies in Response to SCF Volumeof CM Colony #/100,000 Plasmid Transfected Assayed (μl) cells ± SD V19.8(no insert) 100  0  50  0 V19.8 human SCF¹⁻¹⁶² 100 33 ± 7  50 22 ± 3V19.8 rat SCF¹⁻¹⁶² 100 13 ± 1  50 10

The colonies which grew over the 14 day period are shown in FIG. 31A(magnification 12×). The arrow indicates a typical colony. The coloniesresembled the murine HPP-CFC colonies in their large size (average 0.5mm). Due to the presence of EPO, some of the colonies werehemoglobinized. When the colonies were isolated and centrifuged ontoglass slides using a Cytospin (Shandon) followed by staining withWright-Giemsa, the predominant cell type was an undifferentiated cellwith a large nucleus:cytoplasm ratio as shown in FIG. 31B (magnification400×). The arrows in FIG. 31B point to the following structures: arrow1, cytoplasm; arrow 2, nucleus; arrow 3, vacuoles. Immature cells as aclass are large and the cells become progressively smaller as theymature [Diggs et al., The Morphology of Human Blood Cells, Abbott Labs,3 (1978)]. The nuclei of early cells of the hemotopoietic maturationsequence are relatively large in relation to the cytoplasm. In addition,the cytoplasm of immature cells stains darker with Wright-Giemsa thandoes the nucleus. As cells mature, the nucleus stains darker than thecytoplasm. The morphology of the human bone marrow cells resulting fromculture with recombinant human SCF is consistent with the conclusionthat the target and immediate product of SCF action is a relativelyimmature hematopoietic progenitor.

Recombinant human SCF was tested in agar colony assays on human bonemarrow in combination with other growth factors as described above. Theresults are shown in Table 13. SCF synergizes with G-CSF, GM-CSF, IL-3,and EPO to increase the proliferation of bone marrow targets for theindividual CSFs.

TABLE 13 Recombinant human SCF Synergy with Other Human ColonyStimulating Factors Colony #/10⁵ cells (14 Days) mock 0 hG-CSF 32 ± 3hG-CSF + hSCF 74 ± 1 hGM-CSF 14 ± 2 hGM-CSF + hSCF 108 ± 5  hIL-3 23 ± 1hIL-3 + hSCF 108 ± 3  hEPO 10 ± 5 hEPO + IL-3 17 ± 1 hEPO + hSCF  86 ±10 hSCF 0

Another activity of recombinant human SCF is the ability to causeproliferation in soft agar of the human acute myelogenous leukemia (AML)cell line, KG-1 (ATCC CCL 246). COS-1 supernatants from transfectedcells were tested in a KG-1 agar cloning assay [Koeffler et al.,Science, 200, 1153-1154 (1978)] essentially as described except cellswere plated at 3000/ml. The data from triplicate cultures are given inTable 14.

TABLE 14 KG-1 Soft Agar Cloning Assay Volume Colony #/3000 PlasmidTransfected Assayed (μl) Cells ± SD V19.8 (no insert) 25 2 ± 1 V19.8human SCF¹⁻¹⁶² 25 14 ± 0  12 8 ± 0 6 9 ± 5 3 6 ± 4 1.5 6 ± 6 V19.8 ratSCF¹⁻¹⁶² 25 6 ± 1 human GM-CSF 50 (5 ng/ml) 14 ± 5 

B. UT-3 ³H-Thymidine Uptake Assay

UT-7 cells are a human megakaryocyte, huGM-CSF responsive cell lineobtained from John Adamson, New York Blood Center, New York, N.Y. UT-7cells were cultured in Iscove's Modified Dulbecco's Medium, 10% FBS, 1×glutamine, 5 g/ml huGM-CSF. Cells are passaged twice a week at 1×10⁵cells/ml.

Cells were washed twice in phosphate buffered saline (PBS) andresuspended in RPMI medium with 4% FBS and glutamine penicillinstreptomycin (GPS) (Irvine Scientific Cat No. 9316 used at 1% volume pervolume) at 4×10⁴ cells/ml before use. Human SCF along with specificsamples were added to 4000 cells/well in 96 well plates and werecultured for 72 hrs. 0.5 uCi/well of ³H-Thymidine was then added to eachplate, plates were harvested and counted 4 hours later. A typical assayis shown in FIG. 31C.

Activity of human [Met⁻¹]SCF¹⁻¹⁶⁴ and human [Met⁻¹]SCF¹⁻¹⁶⁵, preparedfrom E. coli as described in Example 10, are also equally active instimulating the proliferation of the UT-7 cell line, as shown in FIG.31C.

C. SCF Radio-Receptor Assay Protocol

OCIM1 cells, [Papayannopoulou et al., Blood 72:1029-1038 (1988)] are ahuman erythroleukemic cell line expressing many human SCF receptors percell. These cells are grown in Iscove's Modified Dulbecco's Medium, 10%FBS, and 1× glutamine and passaged 3 times a week to 1×10⁵ cells/ml.

Preparation of the OCIM1 plasma membrane is as follows with all stepsperformed on ice.

First, 40 T175 flasks of cells were grown-up in OCIM1 culture medium,for a total of 1.9×10⁹ cells/ml. The conditioned medium and 1 mM PhenylMethyl Sulfonyl Fluoride (PMSF) protease inhibitor, was spun down in8×250 ml tubes at 1000 rpm for 10 minutes at 4° C. Cells were washedwith PBS and repelleted in 4×50 ml centrifuge tubes at 1000 rpm for 10minutes at 4° C. Cells were resuspended in 20 ml ice cold PBS withglucose sodium pyruvate (Gibco Cat #310-4287). The 20 ml cell solutionwas put into a pre-pressurized, pre-chilled (4° C.) “cell bomb” designedto lyse the cells. Cells were pressurized at 400-650 PSI for 10 minutesto establish equilibrium. When the pressure is released cell lysisoccurs.

At this point the cells were checked for the percentage of cell lysis.90% lysis was common. The cell suspension was resuspended in 80 mlssucrose buffer (0.25 M sucrose, 10 mM Tris, 1 mM EDTA in doubledistilled (dd) H₂O, filtered through a 0.45 u filter, pH 7.0) anddivided between two 40 ml screwcap tubes. Tubes were spun at 5900 RPMfor 10 minutes in a Beckman J2-21 centrifuge, JA-20 rotor at 4° C. Thesupernatants were saved and spun one more time as above to furtherremove any unwanted material. Supernatants were saved and distributedequally into 2 nalgene 40 ml centrifuge tubes. These supernatants werecentrifuged at 16,000 RPM 4° C. for 30 min. in J2-21 centrifuge, JA-20rotor. These supernatants were discarded being careful to save pellets.Each pellet was resuspended in sucrose buffer so there were 20 mls pertube in 4×36 ml plastic ultracentrifuge tubes. Using a 20 ml syringe anda large trochar, the solution was carefully underlayered in each tubewith ice cold 36% sucrose solution (36.1 g sucrose/100 mls ddH₂O),bringing the level of the liquid to within 2 mm of the top of the tube.Without disturbing the interface, each tube was carefully placed intoeach of 6 titanium ultracentrifuge tubes. Tubes were centrifuged at27,000 RPM, 4° C. for 75 minutes in an ultracentrifuge. These tubes werecarefully removed from the rotor and from titanium buckets, placed in arack with the 36% sucrose interface visible. The membraneous material atthe interface was collected with a pasteur pipet and transfered into 2clean nalgene 40 ml centrifuge tubes. Volume was brought up to 40 mlswith ice cold sucrose buffer. Tubes were balanced and centrifuged asbefore at 5900 RPM in J2-21 centrifuge. The supernatant was discardedand each pellet was resuspended in 4 mls ice cold Tris buffer (10 mMTris, 1 mM EDTA, pH 7.0 in ddH₂) with a 1 ml micropipet repeatedly, toensure homogeneity of the solutions. Storage was in 50 ul aliquots at−70° C. in freezing vials.

The SCF radioreceptor assay was conducted as follows with all stepsbeing performed on ice. Human SCF samples were diluted in RRA buffer (50mM Tris, 0.25% BSA pH 7.5) and added to 1.5 ml eppendorf tubes up to 150ul total volume. 50,000 counts in 50 ul buffer of ¹²⁵I—huSCF (providedby ICN radiochemicals) were added to each tube. A dilution of isolatedOCIMl plasma membrane in 50 ul buffer known to give 20% specific bindingwas then added to each tube. Tubes were vortexed and allowed to incubatefor 24 hrs at 4° C. 400 ul of buffer was then added to each tube and thetubes were centrifuged for 8 minutes at 18,000 RPM in J2-21 centrifuge,JA-18.1 fixed angle (45%) rotor, 4° C. All tubes were oriented with lidopening tabs straight up. Supernatants were carefully aspirated by asliding a 21 gauge needle down the side opposite the pellet (hinge sideof tube) to bottom of each tube. Tubes were counted in gamma counter for1 min. each.

In the radioreceptor assay, human [Met⁻¹]SCF¹⁻¹⁶⁴ and human[Met⁻¹]SCF¹⁻¹⁶⁵, prepared from E. coli as described in Example 10,compete equally well with the binding of human [¹²⁵I][[Met⁻¹]SCF¹⁻¹⁶⁴,indicating that they bind equally well to the SCF receptor.

EXAMPLE 10

Purification of Recombinant SCF Products Expressed in E. coli

A. SCF¹⁻¹⁶⁴

Fermentation of E. coli human SCF¹⁻¹⁶⁴ was performed according toExample 6C. The harvested cells (912 g wet weight) were suspended inwater to a volume of 4.6 L and broken by three passes through alaboratory homogenizer (Gaulin Model 15MR-8TBA) at 8000 psi. A brokencell pellet fraction was obtained by centrifugation (17700×g, 30 min, 4°C.), washed once with water (resuspension and recentrifugation), andfinally suspended in water to a volume of 400 ml.

The pellet fraction containing insoluble SCF (estimate of 10-12 g SCF)was added to 3950 ml of an appropriate mixture such that the finalconcentrations of components in the mixture were 8 M urea (ultrapuregrade), 0.1 mM EDTA, 50 mM sodium acetate, pH 6-7; SCF concentration wasestimated as 1.5 mg/ml. Incubation was carried out at room temperaturefor 4 h to solubilize the SCF. Remaining insoluble material was removedby centrifugation (17700×g, 30 min, room temperature). Forrefolding/reoxidation of the solubilized SCF, the supernatant fractionwas added slowly, with stirring, to 39.15 L of an appropriate mixturesuch that the final concentrations of components in the mixture were 2.5M urea (ultrapure grade), 0.01 mM EDTA, 5 mM sodium acetate, 50 mMTris—HCl pH 8.5, 1 mM glutathione, 0.02% (wt/vol) sodium azide. SCFconcentration was estimated at 150 μg/ml. After 60 h at room temperature[shorter times (e.g. ˜20 h) are suitable also], with stirring, themixture was concentrated two-fold using a Millipore Pelliconuntrafiltration apparatus with three 10,000 molecular weight cutoffpolysulfone membrane cassettes (15 ft² total area) and then diafilteredagainst 7 volumes of 20 mM Tris—HCl, pH 8. The temperature during theconcentration/ultrafiltration was 4° C., pumping rate was 5 L/min, andfiltration rate was 600 ml/min. The final volume of recovered retentatewas 26.5 L. By the use of SDS—PAGE carried out both with and withoutreduction of samples, it is evident that most (>80%) of the pelletfraction SCF is solubilized by the incubation with 8 M urea, and thatafter the folding/oxidation multiple species (forms) of SCF are present,as visualized by the SDS—PAGE of unreduced samples. The major form,which represents correctly oxidized SCF (see below), migrates withapparent M_(r) of about 17,000 (unreduced) relative to the molecularweight markers (reduced) described for FIG. 9. Other forms includematerial migrating with apparent M_(r) of about 18-20,000 (unreduced),though to represent SCF with incorrect intrachain disulfide bonds; andbands migrating with apparent M_(r)s in the range of 37,000 (unreduced),or greater, thought to represent various SCF forms having interchaindisulfide bonds resulting in SCF polypeptide chains that are covalentlylinked to form dimers or larger oligomers, respectively. The followingfractionation steps result in removal of remaining E. coli contaminantsand of the unwanted SCF forms, such that SCF purified to apparenthomogeneity, in biologically active conformation, is obtained.

The pH of the ultrafiltration retentate was adjusted to 4.5 by additionof 375 ml of 10% (vol/vol) acetic acid, leading to the presence ofvisible precipitated material. After 60 min, at which point much of theprecipitated material had settled to the bottom of the vessel, the upper24 L were decanted and filtered through a Cuno™ 30SP depth filter at 500ml/min to complete the clarification. The filtrate was then diluted1.5-fold with water and applied at 4° C. to an S-Sepharose Fast Flow(Pharmacia) column (9×18.5 cm) equilibrated in 25 mM sodium acetate, pH4.5. The column was run at a flow rate of 5 L/h, at 4° C. After sampleapplication, the column was washed with five column volumes (˜6 L) ofcolumn buffer and SCF material, which was bound to the column, waseluted with a gradient of 0 to 0.35 M NaCl in column buffer. Totalgradient volume was 20 L and fractions of 200 ml were collected. Theelution profile is depicted in FIG. 33. Aliquots (10 μl) from fractionscollected from the S-Sepharose column were analyzed by SDS—PAGE carriedout both with (FIG. 32A) and without (FIG. 32B) reduction of thesamples. From such analyses it is apparent that virtually all of theabsorbance at 280 nm (FIGS. 32 and 33) is due to SCF material.

The correctly oxidized form predominates in the major absorbance peak(fractions 22-38, FIG. 33). Minor species (forms) which can bevisualized in fractions include the incorrectly oxidized material withapparent M_(r) of 18-20,000 on SDS—PAGE (unreduced), present in theleading shoulder of the main absorbance peak (fractions 10-21, FIG.32B); and disulfide-linked dimer material present throughout theabsorbance region (fractions 10-38, FIG. 32B).

Fractions 22-38 from the S-Sepharose column were pooled, and the poolwas adjusted to pH 2.2 by addition of about 11 ml 6 N HCl and applied toa Vydac C₄ column (height 8.4 cm, diameter 9 cm) equilibrated with 50%(vol/vol) ethanol, 12.5 mM HCl (solution A) and operated at 4° C. Thecolumn resin was prepared by suspending the dry resin in 80% (vol/vol)ethanol, 12.5 mM HCl (solution B) and then equilibrating it withsolution A. Prior to sample application, a blank gradient from solutionA to solution B (6 L total volume) was applied and the column was thenre-equilibrated with solution A. After sample application, the columnwas washed with 2.5 L of solution A and SCF material, bound to thecolumn, was eluted with a gradient from solution A to solution B (18 Ltotal volume) at a flow rate of 2670 ml/h. 286 fractions of 50 ml eachwere collected, and aliquots were analyzed by absorbance at 280 nm (FIG.35), and by SDS—PAGE (25 μl per fraction) as described above (FIG. 34A,reducing conditions; FIG. 34B, nonreducing conditions). Fractions62-161, containing correctly oxidized SCF in a highly purified state,were pooled [the relatively small amounts of incorrectly oxidizedmonomer with M_(r) of about 18-20,000 (unreduced) eluted later in thegradient (about fractions 166-211) and disulfide-linked dimer materialalso eluted later (about fractions 199-235) (FIG. 35)].

To remove ethanol from the pool of fractions 62-161, and to concentratethe SCF, the following procedure utilizing Q-Sepharose Fast Flow(Pharamcia) ion exchange resin was employed. The pool (5 L) was dilutedwith water to a volume of 15.625 L, bringing the ethanol concentrationto about 20% (vol/vol). Then 1 M Tris base (135 ml) was added to bringthe pH to 8, followed by 1 M Tris—HCl, pH 8, (23.6 ml) to bring thetotal Tris concentration to 10 mM. Next 10 mM Tris—HCl, pH 8 (˜15.5 L)was added to bring the total volume to 31.25 L and the ethanolconcentration to about 10% (vol/vol). The material was then applied at4° C. to a column of Q-Sepharose Fast Flow (height 6.5 cm, diameter 7cm) equilibrated with 10 mM Tris—HCl, pH 8, and this was followed bywashing of the column with 2.5 L of column buffer. Flow rate duringsample application and wash was about 5.5 L/h. To elute the bound SCF,200 mM NaCl, 10 mM Tris—HCl, pH 8 was pumped in reverse directionthrough the column at about 200 ml/h. Fractions of about 12 ml werecollected and analyzed by absorbance at 280 nm, and SDS—PAGE as above.Fractions 16-28 were pooled (157 ml).

The pool containing SCF was then applied in two separate chromatographicruns (78.5 ml applied for each) to a Sephacryl S-200 HR (Pharmacia) gelfiltration column (5×138 cm) equilibrated with phosphate-buffered salineat 4° C. Fractions of about 15 ml were collected at a flow rate of about75 ml/h. In each case a major peak of material with absorbance at 280 nmeluted in fractions corresponding roughly to the elution volume range of1370 to 1635 ml. The fractions representing the absorbance peaks fromthe two column runs were combined into a single pool of 525 ml,containing about 2.3 g of SCF. This material was sterilized byfiltration using a Millipore Millipak 20 membrane cartridge.

Alternatively, material from the C₄ column can be concentrated byultrafiltration and the buffer exchanged by diafiltration, prior tosterile filtration.

The isolated recombinant human SCF¹⁻¹⁶⁴ material is highly pure (>98% bySDS—PAGE with silver-staining) and is considered to be a pharmaceuticalgrade. Using the methods outlined in Example 2, it is found that thematerial has amino acid composition and amino acid sequence matchingthose expected from analysis of the SCF gene. The N-terminal amino acidsequence is Met—Glu—Gly—Ile . . . , i.e., the initiating Met residue isretained.

By procedures comparable to those outlined for human SCF¹⁻¹⁶⁴ expressedin E. coli, rat SCF¹⁻¹⁶⁴ (also present in insoluble form inside the cellafter fermention) can be recovered in a purified state with highbiological specific activity. Similarly, human SCF¹⁻¹⁸³ and rat SCF¹⁻¹⁹³can be recovered. The rat SCF¹⁻¹⁹³, during folding/oxidation, tends toform more variously oxidized species, and the unwanted species are moredifficult to remove chromatographically.

The rat SCF¹⁻¹⁹³ and human SCF¹⁻¹⁸³ are prone to proteolytic degradationduring the early stages of recovery, i.e., solubilization andfolding/oxidation. A primary site of proteolysis is located betweenresidues 160 and 170. The proteolysis can be minimized by appropriatemanipulation of conditions (e.g., SCF concentration; varying pH;inclusion of EDTA at 2-5 mM, or other protease inhibitors), and degradedforms to the extend that they are present can be removed by appropriatefractionation steps.

While the use of urea for solubilization, and during folding/oxidation,as outlined, is a preferred embodiment, other solubilizing agents suchas guanidine-HCl (e.g. 6 M during solubilization and 1.25 M duringfolding/oxidation) and sodium N-lauroyl sarcosine can be utilizedeffectively. Upon removal of the agents after folding/oxidation,purified SCFs, as determined by SDS—PAGE, can be recovered with the useof appropriate fractionation steps.

In addition, while the use of glutathione at 1 mM duringfolding/oxidation is a preferred embodiment, other conditions can beutilized with equal or nearly equal effectiveness. These include, forexample, the use in place of 1 mM glutathione of 2 mM glutathione plus0.2 mM oxidized glutathione, or 4 mM glutathione plus 0.4 mM oxidizedglutathione, or 1 mM 2-mercaptoethanol, or other thiol reagents also.

In addition to the chromatographic procedures described, otherprocedures which are useful in the recovery of SCFs in a purified activeform include hydrophobic interaction chromatography [e.g., the use ofphenyl-Sepharose (Pharmacia), applying the sample at neutral pH in thepresence of 1.7 M ammonium sulfate and eluting with a gradient ofdecreasing ammonium sulfate]; immobilized metal affinity chromatography[e.g., the use of chelating-Sepharose (Pharmacia) charged with Cu²⁺ ion,applying the sample at near neutral pH in the presence of 1 mM imidazoleand eluting with a gradient of increasing imidazole]; hydroxylapatitechromatography, [applying the sample at neutral pH in the presence of 1mM phosphate and eluting with a gradient of increasing phosphate]; andother procedures apparent to those skilled in the art.

Other forms of human SCF, corresponding to all or part of the openreading frame encoding by amino acids 1-248 in FIG. 42, or correspondingto the open reading frame encoded by alternatively spliced mRNAs thatmay exist (such as that represented by the cDNA sequence in FIG. 44),can also be expressed in E. coli and recovered in purified form byprocedures similar to those described in this Example, and by otherprocedures apparent to those skilled in the art.

The purification and formulation of forms including the so-calledtransmembrane region referred to in Example 16 may involve theutilization of detergents, including non-ionic detergents, and lipids,including phospholipid-containing liposome structures.

B. SCF¹⁻¹⁶⁵

For the purification of human SCF¹⁻¹⁶⁵ expressed in E. coli, thefollowing information is relevant. After harvesting of cells expressingthe human SCF^(1-165,) pharmaceutical grade human SCF¹⁻¹⁶⁵ was recoveredby procedures the same as those described for human SCF¹⁻¹⁶⁵ (above),but with the following modifications. After cell lysis, the homogenatewas diluted to a volume representing twice the volume of the originalcell suspension, with the inclusion of EDTA to 10 mM finalconcentration. Centrifugation was then done using a Sharples AS-16centrifuge at 15,000 rpm and flow rate of 0.5 L/min, to obtain a pelletfraction. This pellet fraction, without washing, was then subjected tothe solubilization with urea, essentially as described for humanSCF¹⁻¹⁶⁴ except that sodium acetate was omitted, the mixture wastitrated to pH 3 using HCl, the estimated SCF concentration was 3.2mg/ml, and incubation was for 1-2 h at room temperature. All subsequentsteps were at room temperature also. For refolding/reoxidation, themixture was then diluted directly, by a factor of 3.2, such that thefinal conditions included the SCF at about 1 mg/ml, 2.5 M urea, 60 mMNaCl, 1 mM glutathione, 50 mM Tris—HCl, with pH at 8.5. After stirringfor 20-24 h, clarification was accomplished by filtration through a CunoZeta Plus 30SP depth filtration device. A 19 ft² filter was used per 100L of mixture to be filtered. Flow rate during filtration was about 2.9L/min. For a 19 ft² filter, washing of the filter with 50 L of 20 mMTris—HCl, pH 8.5 was done. The following description applies to thehandling of fractions derived from 100 L of refolding/reoxidationmixture. The 150 L of filtrate plus wash was concentrated to 50 L byultrafiltration, and diafiltration against 300 L of 20 mM Tris—HCl, pH8.5 was then done. The diafiltered material was then diluted to 150 L byaddition of the Tris buffer. pH was then adjusted to 4.55 using 10%acetic acid, whereupon the mixture became turbid. 2-24 h later,clarification was accomplished by depth filtration using a 19 ft² CunoZeta Plus 10SP filter, pre-washed with 0.1 M sodium chloride, 50 mMsodium acetate, pH 4.5. After the filtration, the filter was washed with50 L of the same sodium chloride/sodium acetate buffer. The resultingfiltrate plus wash (about 200 L) was applied to an S-Sepharose Fast Flow(Pharmacia) column (10 L bed volume; 30 cm diameter) equilibrated with50 mM sodium acetate, 100 mM sodium chloride, pH 4.5. Flow rate was 1.4L/min. After sample application, the column was washed with 100 L of thecolumn buffer, at a flow rate of 1.2 L/min. Elution was carried out witha linear gradient from the starting column buffer to 50 mM sodiumacetate, 300 mM NaCl, pH 4.5 (200 L total gradient volume), at flow rateof 0.65 L/min. The various forms described for the S-Sepharose Fast Flowfractions obtained in preparation of E. coli-derived human SCF¹⁻¹⁶⁴above were present in essentially the same fashion, and pooling offractions was based on the same criteria as described above. The pooledmaterial (about 25 g SCF in about 20-25 L) was adjusted to pH 2.2 using6 N HCl, and loaded onto a C4 column (1.2 L bed volume; 14 cm diameter;Vydac Proteins C₄, Cat. No. 214TPB2030), at 100 ml/min. The column wasnext washed with 10 L of 25% ethanol, 12.5 mM HCl, and theneluted with alinear gradient fromthis buffer to 75% ethanol, 12.5 mM HCl (25 L totalgradient volume). Again, the various species present in the elutedfractions, and the pooling of fractions, were essentially as describedfor the SCF¹⁻¹⁶⁴. The pool, containing about 16 g SCF¹⁻¹⁶⁵correctly-oxidized monomer in a volume of about 9 ml, was diluted6.25-fold, made 10 mM in sodium phosphate by addition of 0.5 M sodiumphosphate, pH 6.5, and titrated to pH 6.5 using 1 N sodium hydroxide.The material was then applied at a flow rate of 400 ml/min to aQ-Sepharose Fast Flow (Pharmacia) column (2 L bed volume; 14 cmdiameter) equilibrated with 10 mM sodium phosphate, pH 6.5. Afterwashing the column with 20 L of 10 mM sodium phosphate, 25 mM sodiumchloride, pH 6.5, elution was carried out with a linear gradient fromthe wash buffer to 10 mM sodium phosphate, 100 mM NaCl, pH 6.5.Fractions corresponding to the main absorbance (at 280 nm) peakrepresent the correctly-oxidized SCF¹⁻¹⁶⁵. These fractions were pooled;typically the pool contained about 12-15 g SCF¹⁻¹⁶⁵, in a volume ofabout 17-18 L. The SCF material was then concentrated by ultrafiltrationand other buffers optionally introduced by diafiltration, a preferredbuffer being 10 mM sodium acetate, 140 mM sodium chloride, pH 5.

C. SCF¹⁻²⁴⁸

The full length recombinant human stem cell factor (SCF¹⁻²⁴⁸) is formedin E. coli as inclusion bodies. After isolation of the inclusion bodies,treatment with 8 M urea, 50 mM sodium acetate, 0.1 mM EDTA, pH 5.0 doesnot solubilize any SCF¹⁻²⁴⁸. This is in contrast to shorter SCFs whichsolubilize well in this buffer. To solubilize SCF¹⁻²⁴⁸, the urea-washedinclusion bodies are suspended in 50 mM Tris—HCl, 1 mM EDTA, 2% sodiumdeoxycholate (NaDOC), pH 8.5 at an approximate SCF¹⁻²⁴⁸ concentration of0.2 to 1.0 mg/mL. To this is added powdered dithiothreitol (DTT) to aconcentration of 20 mM. The mixture is stirred for 2.5 hours at roomtemperature. Unsolubilized debris is removed by centrifuing at 20,000×gfor 20 min. The supernatant contains all of the SCF¹⁻²⁴⁸ which runs as afuzzy 33,000 dalton band on a reducing SDS polyacrylamide gel. BothNaDOC, an anionic detergent, and DTT, a reducing agent are required forsolubilization.

Soluble oxidized SCF¹⁻²⁴⁸ can be prepared by diluting the solubilizationmixture supernatant with nine volumes of 50 mM Tris, 1 mM EDTA, 2% NaDOC(no pH adjustment). The pH of the diluted mixture is approximately 9.5.This mixture is stirred vigorously at room temperature for approximately40 hours. This mixture can be clarified by filtration through a 0.45μcellulose acetate membrane. The filtrate contains SCF¹⁻²⁴⁸ which runs asa 28,000 dalton band on a non-reducing SDS polyacrylamide gel. Underreducing conditions, the fuzzy 33,000 dalton band is visible. Thefiltrate also contains smaller but variable amounts of incompletelyoxidized SCF¹⁻²⁴⁸ and an apparent disulfide-linked dimer atapproximately 80,000 daltons on the gels. Upon removal of NaDOC bydiafiltration using a 10,000 dalton molecular weight cut-off membrane,the oxidized SCF¹⁻²⁴⁸ remains in solution.

SCF¹⁻²⁴⁸ was subsequently purified to 80-90% purity by a combination ofanion exchange, gel filtration, and cation exchange chromatography. Theprotein requires the presence of the non-ionic detergent, Triton X-100,to remain unaggregated. Material following anion exchange chromatographywas active in the UT-7 assay (Example 9B). The final material aftercation exchange chromatography showed no activity in the UT-7 assay. Itmay be that earlier samples contained some active proteolyzed SCF. TheSCF¹⁻²⁴⁸ diluted in detergent-free buffer for assay may be incapable ofinteraction with the SCF receptor because of aggregation.

EXAMPLE 11

Recombinant SCF from Mammalian Cells

A. Fermentation of CHO Cells Producing SCF

Recombinant Chinese hamster ovary (CHO) cells (strain CHO pDSRα2hSCF¹⁻¹⁶²) were grown on microcarriers in a 20 liter perfusion culturesystem for the production of human SCF¹⁻¹⁶². The fermentor system issimilar to that used for the culture of BRL 3A cells, Example 1B, exceptfor the following: The growth medium used for the culture of CHO cellswas a mixture of Dulbecco's Modified Eagle Medium (DMEM) and Ham's F-12nutrient mixture in a 1:1 proportion (GIBCO), supplemented with 2 mMglutamine, nonessential amino acids (to double the existingconcentration by using 1:100 dilution of Gibco #320-1140) and 5% fetalbovine serum. The harvest medium was identical except for the omissionof serum. The reactor was inoculated with 5.6×10⁹ CHO cells grown in two3-liter spinner flasks. The cells were allowed to grow to aconcentration of 4×10⁵ cells/ml. At this point 100 grams ofpresterilized cytodex-2 microcarriers (Pharmacia) were added to thereactor as a 3-liter suspension in phosphate buffered saline. The cellswere allowed to attach and grow on the microcarriers for four days.Growth medium was perfused through the reactor as needed based onglucose consumption. The glucose concentration was maintained atapproximately 2.0 g/L. After four days, the reactor was perfused withsix volumes of serum-free medium to remove most of the serum (proteinconcentration <50 μg/ml). The reactor was then operated batch-wise untilthe glucose concentration fell below 2 g/L. From this point onward, thereactor was operated at a continuous perfusion rate of approximately 20L/day. The pH of the culture was maintained at 6.9±0.3 by adjusting theCO₂ flow rate. The dissolved oxygen was maintained higher than 20% ofair saturation by supplementing with pure oxygen as necessary. Thetemperature was maintained at 37±0.5° C.

Approximately 450 liters of serum-free conditioned medium was generatedfrom the above system and was used as starting material for thepurification of recombinant human SCF¹⁻¹⁶².

Approximately 589 liters of serum-free conditioned medium was alsogenerated in similar fashion but using strain CHO pDSRα2 rSCF¹⁻¹⁶² andused as starting material for purification of rat SCF¹⁻¹⁶².

B. Purification of Recombinant Mammalian Expressed Rat SCF¹⁻¹⁶² andOther Recombinant Mammalian SCFs

All purification work was carried out at 4° C. unless indicatedotherwise.

1. Concentration and Diafiltration

Conditioned medium generated by serum-free growth of cell strain CHOpDSRα2 rat SCF¹⁻¹⁶² as performed in Section A above, was clarified byfiltration thru 0.45μ Sartocapsules (Sartorius). Several differentbatches (36 L, 101 L, 102 L, 200 L and 150 L) were separately subjectedto concentration and diafiltration/buffer exchange. To illustrate, thehandling of the 36 L batch was as follows. The filtered condition mediumwas concentrated to ˜500 ml using a Millipore Pellicon tangential flowultrafiltration apparatus with three 10,000 molecular weight cutoffcellulose acetate membrane cassettes (15 ft² total membrane area; pumprate ˜2,200 ml/min and filtration rate ˜750 ml/min).Diafiltration/buffer exchange in preparation for anion exchangechromatography was then accomplished by adding 1000 ml of 10 mMTris—HCl, pH 6.7-6.8 to the concentrate, reconcentrating to 500 ml usingthe tangential flow ultrafiltration apparatus, and repeating this 5additional times. The concentrated/diafiltered preparation was finallyrecovered in a volume of 1000 ml. The behavior of all conditioned mediumbatches subjected to the concentration and diafiltration/buffer exchangewas similar. Protein concentrations for the batches, determined by themethod of Bradford [Anal. Bioch. 72, 248-254 (1976)] with bovine serumalbumin as standard, were in the range 70-90 μg/ml. The total volume ofconditioned medium utilized for this preparation was about 589 L.

2. Q-Sepharose Fast Flow Anion Exchange Chromatography

The concentrated/diafiltered preparations from each of the fiveconditioned medium batches referred to above were combined (total volume5,000 ml). pH was adjusted to 6.75 by adding 1 M HCl. 2000 ml of 10 mMTris—HCl, pH 6.7 was used to bring conductivity to about 0.700 mmho. Thepreparation was applied to a Q-Sepharose Fast Flow anion exchange column(36×14 cm; Pharmacia Q-Sepharose Fast Flow resin) which had beenequilibrated with the 10 mM Tris—HCl, pH 6.7 buffer. After sampleapplication, the column was washed with 28,700 ml of the Tris buffer.Following this washing the column was washed with 23,000 ml of 5 mMacetic acid/1 mM glycine/6 M urea/20 μM CuSO₄ at about pH 4.5. Thecolumn was then washed with 10 mM Tris—HCl, 20 μm CuSo₄, pH 6.7 bufferto return to neutral pH and remove urea, and a salt gradient (0-700 mMNaCl in the 10 mM Tris—HCl, 20 μM CuSO₄, pH 6.7 buffer; 40 L totalvolume) was applied. Fractions of about 490 ml were collected at a flowrate of about 3,250 ml/h. The chromatogram is shown in FIG. 36. “MC/9cpm” refers to biological activity in the MC/9 assay; 5 μl from theindicated fractions was assayed. Eluates collected during the sampleapplication and washes are not shown in the Figure; no biologicalactivity was detected in these fractions.

3. Chromatography Using Silica-Bound Hydrocarbon Resin

Fractions 44-66 from the run shown in FIG. 36 were combined (11,200 ml)and EDTA was added to a final concentration of 1 mM. This material wasapplied at a flow rate of about 2000 ml/h to a C₄ column (Vydac ProteinsC₄; 7×8 cm) equilibrated with buffer A (10 mM Tris pH 6.7/20% ethanol).After sample application the column was washed with 1000 ml of buffer A.A linear gradient from buffer A to buffer B (10 mM Tris pH 6.7/94%ethanol) (total volume 6000 ml) was then applied, and fractions of 30-50ml were collected. Portions of the C₄ column starting sample, runthroughpool and wash pool in addition to 0.5 ml aliquots of the gradientfractions were dialyzed against phosphate-buffered saline in preparationfor biological assay. These various fractions were assayed by the MC/9assay (5 μl aliquots of the prepared gradient fractions; cmp in FIG.37). SDS—PAGE [Laemmli, Nature 227, 680-685 (1970); stacking gelscontained 4% (w/v) acrylamide and separating gels contained 12.5% (w/v)acrylamide] of aliquots of various fractions is shown in FIG. 38. Forthe gels shown, sample aliquots (100 μl) were dried under vacuum andthen redissolved using 20 μl sample treatment buffer (reducing, i.e.,with 2-mercaptoethanol) and boiled for 5 min prior to loading onto thegel. The numbered marks at the left of the Figure represent migrationpositions of molecular weight markers (reduced) as in FIG. 6. Thenumbered lanes represent the corresponding fractions collected duringapplication of the last part of the gradient. The gels weresilver-stained [Morrissey, Anal. Bioch. 117, 307-310 (1981)].

4. Q-Sepharose Fast Flow Anion Exchange Chromatography

Fractions 98-124 from the C₄ column shown in FIG. 37 were pooled (1050ml). The pool was diluted 1:1 with 10 mM Tris, pH 6.7 buffer to reduceethanol concentration. The diluted pool was then applied to aQ-Sepharose Fast Flow anion exchange column (3.2×3 cm, PharmaciaQ-Sepharose Fast Flow resin) which had been equilibrated with the 10 mMTris—HCl, pH 6.7 buffer. Flow rate was 463 ml/h. After sampleapplication the column was washed with 135 ml of column buffer andelution of bound material was carried out by washing with 10 mMTris—HCl, 350 mM NaCl, pH 6.7. The flow direction of the column wasreversed in order to minimize volume of eluted material, and 7.8 mlfractions were collected during elution.

5. Sephacryl S-200 HR Gel Filtration Chromatography

Fractions containing eluted protein from the salt wash of theQ-Sepharose Fast Flow anion exchange column were pooled (31 ml). 30 mlwas applied to a Sephacryl S-200 HR (Pharmacia) gel filtration column,(5×55.5 cm) equilibrated in phosphate-buffered saline. Fractions of 6.8ml were collected at a flow rate of 68 ml/hr. Fractions corresponding tothe peak of absorbance at 280 nm were pooled and represent the finalpurified material.

Table 15 shows a summary of the purification.

TABLE 15 Summary of Purification of Mammalian Expressed Rat SCF¹⁻¹⁶²Total Step Volume (ml) Protein (mg)* Conditioned medium (concentrated)7,000 28,420 Q-Sepharose Fast Flow 11,200 974 C₄ resin 1,050 19Q-Sepharose Fast Flow 31 20 Sephacryl S-200 HR 82 19** *Determined bythe method of Bradford (supra, 1976). **Determined as 47.3 mg byquantitative amino acid analysis using methodology similar to thatoutlined in Example 2.

The N-terminal amino acid sequence of purified rat SCF¹⁻¹⁶² isapproximately half Gln—Glu—Ile . . . and half PyroGlu—Glu—Ile . . . , asdetermined by the methods outlined in Example 2. This result indicatesthat rat SCF¹⁻¹⁶² is the product of proteolytic processing/cleavagebetween the residues indicated as numbers (−1) (Thr) and )+1) (Gln) inFIG. 14C. Similarly, purified human SCF¹⁻¹⁶² from transfected CHO cellconditioned medium (below) has N-terminal amino acid sequenceGlu—Gly—Ile, indicating that it is the product of processing/cleavagebetween residues indicated as numbers (−1) (Thr) and (+1) (Glu) in FIG.15C.

Using the above-described protocol will yield purified human SCFprotein, either recombinant forms expressed in CHO cells or naturallyderived.

Additional purification methods that are of utility in the purificationof mammalian cell derived recombinant SCFs include those outlined inExamples 1 and 10, and other methods apparent to those skilled in theart.

Other forms of human SCF, corresponding to all or part of the openreading frame encoded by amino acids 1-248 shown in FIG. 42, orcorresponding to the open reading frame encoded by alternatively splicedmRNAs that may exist (such as that represented by the cDNA sequence inFIG. 44), can also be expressed in mammalian cells and recovered inpurified form by procedures similar to those described in this Example,and by other procedures apparent to those skilled in the art.

C. SDS—PAGE and Glycosidase Treatments

SDS—PAGE of pooled fractions from the Sephacryl S-200 HR gel filtrationcolumn is shown in FIG. 39; 2.5 μl of the pool was loaded (lane 1). Thelane was silver-strained. Molecular weight markers (lane 6) were asdescribed in FIG. 6. The different migrating material above and slightlybelow the M_(r) 31,000 marker position represents the biologicallyactive material; the apparent heterogeneity is largely due to theheterogeneity in glycosylation.

To characterize the glycosylation purified material was treated with avariety of glycosidases, analyzed by SDS—PAGE (reducing conditions) andvisualized by silver-staining. Results are shown in FIG. 39. Lane 2,neuraminidase. Lane 3, neuraminidase and O-glycanase. Lane 4,neuraminidase, O-glycanase and N-glycanase. Lane 5, neuraminidase andN-glycanase. Lane 7, N-glycanase. Lane 8, N-glycanase without substrate.Lane 9, O-glycanase without substrate. Conditions were 10 mM3-[(3-cholamidopropyl) dimethyl ammonio]-1-propane sulfonate (CHAPS),66.6 mM 2-mercaptoethanol, 0.04% (wt/vol) sodium azide, phosphatebuffered saline, for 30 min at 37° C., followed by incubation at half ofdescribed concentration in presence of glycosidases for 18 h at 37° C.Neuraminidase (from Arthrobacter ureafaciens; supplied by Calbiochem)was used at 0.5 units/ml final concentration. O-Glycanase (Genzyme;endo-alpha-N-acetyl galactosaminidase) was used at 7.5 milliunits/ml.N-Glycanase (Genzyme; peptide: N-glycosidase F;peptide-N⁴[N-acetyl-beta-glucosaminyl] asparagine amidase) was used at10 units/ml.

Where appropriate, various control incubations were carried out. Theseincluded: incubation without glycosidase, to verify that results weredue to the glycosidase preparations added; incubation with glycosylatedproteins (e.g. glycosylated recombinant human erythropoietin) known tobe substrates for the glycosidases, to verify that the glycosidaseenzymes used were active; and incubation with glycosidases but nosubstrate, to judge where the glycosidase preparations were contributingto or obscuring the visualized gel bands (FIG. 39, lanes 8 and 9).

A number of conclusions can be drawn from the experiments describedabove. The various treatments with N-glycanase [which removes bothcomplex and high-mannose N-linked carbohydrate (Tarentine et al.,Biochemistry 24, 4665-4671 (1988)], neuraminidase (which removes sialicacid residues), and O-glycanase [which removes certain O-linkedcarbohydrates (Lambin et al., Biochem. Soc. Trans. 12, 599-600 (1984)],suggest that: both N-linked and O-linked carbohydrates are present; andsialic acid is present, with at least some of it being part of theO-linked moieties. The fact that treatment with N-glycanase can convertthe heterogeneous material apparent by SDS-PAGE to a faster-migratingform which is much more homogeneous indicates that all of the materialrepresents the same polypeptide, with the heterogeneity being causedmainly by heterogeneity in glycosylation.

While the results of this section apply to purified CHO cell-derived ratSCF¹⁻¹⁶², equivalent results of SDS-PAGE and glycosidase treatments areobtained for CHO cell-derived human SCF¹⁻¹⁶².

EXAMPLE 12 Preparation of Recombinant SCF PEG

A. Preparation of Recombinant SCF¹⁻¹⁶⁴ PEG

Rat SCF¹⁻¹⁶⁴, purified from a recombinant E. coli expression systemaccording to Examples 6A and 10, was used as starting material forpolyethylene glycol modification described below.

Methoxypolyethylene glycol-succinimidyl succinate (18.1 mg=3.63 umol;SS-MPEG=Sigma Chemical Co. no. M3152, approximate molecularweight=5,000) in 0.327 mL deionized water was added to 13.3 mg (0.727umol) recombinant rat SCF¹⁻¹⁶⁴ in 1.0 mL 138 mM sodium phosphate, 62 mMNaCl, 0.62 mM sodium acetate, pH 8.0. The resulting solution was shakengently (100 rpm) at room temperature for 30 minutes. A 1.0 mL aliquot ofthe final reaction mixture (10 mg protein) was then applied to aPharmacia Superdex 75 gel filtration column (1.6×50 cm) and eluted with100 mM sodium phosphate, pH 6.9, at a rate of 0.25 mL/min at roomtemperature. The first 10 mL of column effluent were discarded, and 1.0mL fractions were collected thereafter. The UV absorbance (280 nm) ofthe column effluent was monitored continuously and is shown in FIG. 40A.Fractions number 25 through 27 were combined and sterilized byultrafiltration through a 0.2μ polysulfone membrane (Gelman Sciences no.4454), and the resulting pool was designated PEG-25. Likewise, fractionsnumber 28 through 32 were combined, sterilized by ultrafiltration, anddesignated PEG-32. Pooled fraction PEG-25 contained 3.06 mg protein andpooled fraction PEG-32 contained 3.55 mg protein, as calculated fromA280 measurements using for calibration an absorbance of 0.66 for a 1.0mg/ml solution of unmodified rat SCF¹⁻¹⁶⁴. Unreacted rat SCF¹⁻¹⁶⁴,representing 11.8% of the tool protein in the reaction mixture, waseluted in fractions number 34 to 37. Under similar chromatographicconditions, unmodified rat SCF¹⁻¹⁶⁴ was eluted as a major peak with aretention volume of 45.6 mL, FIG. 40B. Fractions number 77 to 80 in FIG.40A contained N-hydroxysuccinimide, a by-product of the reaction of ratSCF¹⁻¹⁶⁴ with SS-MPEG.

Potentially reactive amino groups in rat SCF¹⁻¹⁶⁴ include 12 lysineresidues and the alpha amino group of the N-terminal glutamine residue.Pooled fraction PEG-25 contained 9.3 mol of reactive amino groups permol of protein, as determined by spectroscopic titration withtrinitrobenzene sulfonic acid (TNBS) using the method described byHabeeb, Anal. Biochem. 14:328-336 (1966). Likewise, pooled fractionPEG-32 contained 10.4 mol and unmodified rat SCF¹⁻¹⁶⁴ contained 13.7 molof reactive amino groups per mol of protein, respectively. Thus, anaverage of 3.3 (13.7 minus 10.4) amino groups of rat SCF¹⁻¹⁶⁴ in pooledfraction PEG-32 were modified by reaction with SS-MPEG. Similarly, anaverage of 4.4 amino groups of rat SCF¹⁻¹⁶⁴ in pooled fraction PEG-25were modified. Human SCF (hSCF¹⁻¹⁶⁴) produced as in Example 10 was alsomodified using the procedures noted above. Specifically, 714 mg (38.5umol) hSCF¹⁻¹⁶⁴ were reacted with 962.5 mg (192.5 umol) SS-MPEG in 75 mLof 0.1 M sodium phosphate buffer, pH 8.0 for 30 minutes at roomtemperature. The reaction mixture was applied to a Sephacryl S-200HRcolumn (5×134 cm) and eluted with PBS (Gibco Dulbecco'sphosphate-buffered saline without CaCl₂ and MgCl₂) at a rate of 102mL/hr, and 14.3-mL fractions were collected. Fractions no. 39-53,analogous to the PEG-25 pool described above and in FIG. 40A, werepooled and found to contain a total of 354 mg of protein. In vivoactivity of this modified SCF in primates is presented in Example 8C.

B. Preparation of Recombinant SCF¹⁻¹⁶⁵ PEG

Recombinant human SCF¹⁻¹⁶⁵ produced as in Example 10 was coupled tomethoxypolyethylene glycol (MW=6,000) by reacting 334 mg (18.0 μmol) ofrhuSCF¹⁶⁵ with 433 mg (72.2 μmol) of the N-hydroxysuccinimidyl ester ofcarboxymethyl-MPEG [prepared by procedures described by Veronese, F. M.,et al., J. Controlled Release, 10:145-154 (1989) in 33.4 ml of 0.1 Mbicine buffer, pH 8.0 for 1 hour at room temperature. The reactionmixture was diluted with 134 ml of water for injection (WFI), titratedto pH 4.0 with 0.5 N HCl, filtered through a 0.20μ cellulose acetatefilter (Nalgene no. 156-4020), and applied at a rate of 5.0 ml/min to a2.6×19.5 cm column of S-Sepharose FF (Pharmacia) which had beenpreviously equilibrated with 20 mM sodium acetate, pH 4.0 at roomtemperature. Effluent from the column was collected in 8.0-ml fractions(no. 1-18) during sample loading, and the ultraviolet absorbance (A₂₈₀)of the effluent was monitored continuously. The column was thensequentially washed with 200 ml of the equilibration buffer at 5.0ml/min (fractions no. 19-44), with 200 ml of 20 mM sodium acetate, 0.5 MNaCl, pH 4.0 at 8.0 ml/min (fractions no. 45-69), and finally with 200ml of 20 mM sodium acetate, 1.0 M NaCl, pH 4.0 at 8.0 ml/min (fractionsNo. 70-94). Fractions (no. 28-31 and 55-62) containing MPEG-rhu-SCF¹⁻¹⁶⁵were combined and dialyzed by ultrafiltration (Amicon YM-10 membrane)against 10 mM sodium acetate, 140 mM NaCl, pH 5.0 to yield 284 mg offinal product in a volume of 105 ml. The resulting MPEG-rhu-SCF¹⁶⁵ wasshown to be free of unbound MPEG and other reaction by-products byanalytical size-exclusion HPLC [Toso-Haas TSK G3000 SWXL and G4000 SWXLcolumns (each 0.68×30 cm; 5 u) connected in tandem; 0.1 M sodiumphosphate, pH 6.9 at 1.0 ml/min at room temperature; UV absorbance (280nm) and refractive index detectors in series].

EXAMPLE 13 SCF Receptor Expression on Leukemic Blasts

Leukemic blasts were harvested from the peripheral blood of a patientwith a mixed lineage leukemia. The cells were purified by densitygradient centrifugation and adherence depletion. Human SCF¹⁻¹⁶⁴ wasiodinated according to the protocol in Example 7. The cells wereincubated with different concentrations of iodinated SCF as described[Broudy, Blood, 75 1622-1626 (1990)]. The results of the receptorbinding experiment are shown in FIG. 41. The receptor density estimatedis approximately 70,000 receptors/cell.

EXAMPLE 14 Rat SCF Activity on Early Lymphoid Precursors

The ability of recombinant rat SCF¹⁻¹⁶⁴ (rrSCF¹⁻¹⁶⁴), to actsynergistically with IL-7 to enhance lymphoid cell proliferation wasstudied in agar cultures of mouse bone marrow. In this assay, thecolonies formed with rrSCF¹⁻¹⁶⁴ alone contained monocytes, neutrophils,and blast cells, while the colonies stimulated by IL-7 alone or incombination with rrSCF¹⁻¹⁶⁴ contained primarily pre-B cells. Pre-Bcells, characterized as B220⁺, sIg⁻, cμ³⁰, were identified by FACSanalysis of pooled cells using fluorescence-labeled antibodies to theB220 antigen [Coffman, Immunol. Rev., 69, 5 (1982)] and to surface Ig(FITC-goat anti-K, Southern Biotechnology Assoc., Birmingham, Ala.); andby analysis of cytospin slides for cytoplasmic μ expression usingfluorescence-labeled antibodies (TRITC-goat anti-μ, SouthernBiotechnology Assoc.,). Recombinant human IL-7 (rhIL-7) was obtainedfrom Biosource International (Westlake Village, Calif.). When rrSCF¹⁻¹⁶⁴was added in combination with the pre-B cell growth factor IL-7, asynergistic increase in colony formation was observed (Table 16),indicating a stimulatory role of rrSCF¹⁻¹⁶⁴ on early B cell progenitors.

Table 16. Stimulation of Pre-B Cell Colony Formation by rrSCF¹⁻¹⁶⁴ inCombination with hIL-7

Growth Factors Colony Number¹ Saline  0 rrSCF¹⁻¹⁶⁴ 200 ng 13 ± 2 100 ng 7 ± 4 50 ng  4 ± 2 rhIL-7 200 ng 21 ± 6 100 ng 18 ± 6 50 ng 13 ± 6 25ng  4 ± 2 rhIL-7 200 ng + rrSCF¹⁻¹⁶⁴ 200 ng 60 ± 0 100 ng + 200 ng 48 ±8 50 ng + 200 ng 24 ± 10 25 ng + 200 ng 21 ± 2 ¹Number of colonies per 5× 10⁴ mouse bone marrow cells plated.

Each value is the mean of triplicate dishes±SD.

EXAMPLE 15 Identification of the Receptor for SCF

A. c-kit is the Receptor for SCF¹⁻¹⁶⁴

To test whether SCF¹⁻¹⁶⁴ is the ligand for c-kit, the cDNA for theentire murine c-kit [Qiu et al., EMBO J., 7, 1003-1011 (1988)] wasamplified using PCR from the SCF¹⁻¹⁶⁴ responsive mast cell line MC/9[Nabel et al., Nature, 291, 332-334 (1981)] with primers designed fromthe published sequence. The ligand binding and transmembrane domains ofhuman c-kit, encoded by amino acids 1-549 [Yarden et al., EMBO J., 6,3341-3351 (1987)], were cloned using similar techniques from the humanerythroleukemia cell line, HEL [Martin and Papayannopoulou, Science,216, 1233-1235 (1982)]. The c-kit cDNAs were inserted into the mammalianexpression vector V19.8 transfected into COS-1 cells, and membranefractions prepared for binding assays using either rat or human¹²⁵I-SCF¹⁻¹⁶⁴ according to the methods described in Sections B and Cbelow. Table 17 shows the data from a typical binding assay. There wasno detectable specific binding of ¹²⁵I human SCF¹⁻¹⁶⁴ to COS-1 cellstransfected with V19.8 alone. However, COS-1 cells expressing humanrecombinant c-kit ligand binding plus transmembrane domains (hckit-LT1)did bind ¹²⁵I-hSCF¹⁻¹⁶⁴ (Table 17). The addition of a 200 fold molarexcess of unlabelled human SCF¹⁻¹⁶⁴ reduced binding to backgroundlevels. Similarly, COS-1 cells transfected with the full length murinec-kit (mckit-L1) bound rat ¹²⁵I-SCF¹⁻¹⁶⁴. A small amount of rat¹²⁵I-SCF¹⁻¹⁶⁴ binding was detected in COS-1 cells transfectants withV19.8 alone, and has also been observed in untransfected cells (notshown), indicating that COS-1 cells express endogenous c-kit. Thisfinding is in accord with the broad cellular distribution of c-kitexpression. Rat ¹²⁵I-SCF¹⁻¹⁶⁴ binds similarly to both human and murinec-kit, while human ¹²⁵I-SCF¹⁻¹⁶⁴ bind with lower activity to murinec-kit (Table 17). This data is consistent with the pattern of SCF¹⁻¹⁶⁴cross-reactivity between species. Rat SCF¹⁻¹⁶⁴ induces proliferation ofhuman bone marrow with a specific activity similar to that of humanSCF¹⁻¹⁶⁴, while human SCF¹⁻¹⁶⁴ induced proliferation of murine mastcells occurs with a specific activity 800 fold less than the ratprotein.

In summary, these findings confirm that the phenotypic abnormalitiesexpressed by W or S1 mutant mice are the consequences of primary defectsin c-kit receptor/ligand interactions which are critical for thedevelopment of diverse cell types.

TABLE 17 SCF^(1-164 Binding to Recombinant) c-kit Expressed in COS-1Cells Plasmid CPM Bound^(a) Trans- Human SCF¹⁻¹⁶⁴ Rat SCF¹⁻¹⁶⁴ fected¹²⁵I-SCF^(b) ¹²⁵I-SCF + cold^(c) ¹²⁵I-SCF^(d) ¹²⁵I-SCF + cold^(e) V19.82,160 2,150 1,100 550 V19.8: 59,350 2,380 70,000 1,100 hckit-LT1 V19.8:9,500 1,100 52,700 600 mckit-L1 ^(a)The average of duplicatemeasurements is shown; the experiment has been independently performedwith similar results three times. ^(b)1.6 nM human ¹²⁵I-SCF¹⁻¹⁶⁴ ^(c)1.6nM human ¹²⁵I-SCF¹⁻¹⁶⁴ + 320 nM unlabelled human SCF¹⁻¹⁶⁴ ^(d)1.6 nM rat¹²⁵I-SCF¹⁻¹⁶⁴ ^(e)1.6 nM rat ¹²⁵I-SCF¹⁻¹⁶⁴ + 320 nM unlabelled ratSCF¹⁻¹⁶⁴

B. Recombinant c-kit Expression in COS-1 Cells

Human and murine c-kit cDNA clones were derived using PCR techniques[Saiki et al., Science, 239, 487-491 (1988)] from total RNA isolated byan acid phenol/chloroform extraction procedure [Chomzynsky and Sacchi,Anal. Biochem., 162, 156-159, (1987)] from the human erythroleukemiacell line HEL and MC/9 cells, respectively. Unique sequenceoligonucleotides were designed from the published human and murine c-kitsequences. First strand cDNA was synthesized from the total RNAaccording to the protocol provided with the enzyme, Mo-MLV reversetranscription (Bethesda Research Laboratories, Bethesda, Md.), usingc-kit antisense oligonucleotides as primers. Amplification ofoverlapping regions of the c-kit ligand binding and tyrosine kinasedomains was accomplished using appropriate pairs of c-kit primers. Theseregions were cloned into the mammalian expression vector V19.8 (FIG. 17)for expression in COS-1 cells. DNA sequencing of several clones revealedindependent mutations, presumably arising during PCR amplification, inevery clone. A clone free of these mutations was constructed byreassembly of mutation-free restriction fragments from separate clones.Some differences from the published sequence appeared in all or in abouthalf of the clones; these were concluded to be the actual sequencespresent in the cell lines used, and may represent allelic differencesfrom the published sequences. The following plasmids were constructed inV19.8: V19.8:mckit-LT1, the entire murine c-kit; and V19.8:hckit-L1,containing the ligand binding plus transmembrane region (amino acids1-549) of human c-kit.

The plasmids were transfected into COS-1 cells essentially as describedin Example 4.

C. ¹²⁵I-SCF¹⁻¹⁶⁴ Binding to COS-1 Cells Expressing Recombinant c-kit

Two days after transfection, the COS-1 cells were scraped from the dish,washed in PBS, and frozen until use. After thawing, the cells wereresuspended in 10 mM Tris-HCl, 1 mM MgCl₂ containing 1 mM PMSF, 100μg/ml aprotinin, 25 μg/ml leupeptin, 2 μg/ml pepstatin, and 200 μg/mlTLCK-HCl. The suspension was dispersed by pipetting up and down 5 times,incubated on ice for 15 minutes, and the cells were homogenized with15-20 strokes of a Dounce homogenizer. Sucrose (250 mM) was added to thehomogenate, and the nuclear fraction and residual undisrupted cells werepelleted by centrifugation at 500×g for 5 min. The supernatant wascentrifuged at 25,000 g for 30 min. at 4° C. to pellet the remainingcellular debris. Human and rat SCF¹⁻¹⁶⁴ were radioiodinated usingchloramine-T [Hunter and Greenwood, Nature, 194, 495-496 (1962)]. COS-1membrane fractions were incubated with either human or rat ¹²⁵I-SCF¹⁻¹⁶⁴(1.6 nM) with or without a 200 fold molar excess of unlabelled SCF¹⁻¹⁶⁴in binding buffer consisting of RPMI supplemented with 1% bovine serumalbumin and 50 mM HEPES (pH 7.4) for 1 h at 22° C. At the conclusion ofthe binding incubation, the membrane preparations were gently layeredonto 150 μl of phthalate oil and centrifuged for 20 minutes in a BeckmanMicrofuge 11 to separate membrane bound ¹²⁵I-SCF¹⁻¹⁶⁴ from free¹²⁵I-SCF¹⁻¹⁶⁴. The pellets were clipped off and membrane associated¹²⁵I-SCF¹⁻¹⁶⁴ was quantitiated.

EXAMPLE 16 Isolation of a Human SCF cDNA

A. Construction of the HT-1080 cDNA Library

Total RNA was isolated from human fibrosarcoma cell line HT-1080 (ATCCCCL 121) by the acid guanidinium thiocyanate-phenol-chloroformextraction method [Chomczynski et al., Anal. Biochem. 162, 156 (1987)],and poly(A) RNA was recovered by using oligo(dT) spin column purchasedfrom Clontech. Double-stranded cDNA was prepared from 2 μg poly(A) RNAwith a BRL (Bethesda Research Laboratory) cDNA synthesis kit under theconditions recommended by the supplier. Approximately 100 ng of columnfractionated double-stranded cDNA with an average size of 2 kb wasligated to 300 ng SalI/NotI digested vector pSORT 1 [D'Alessio et al.,Focus, 12, 47-50 (1990)] and transformed into DH5α (BRL, Bethesda, Md.)cells by electroporation [Dower et al., Nucl. Acids Res., 16, 6127-6145(1988)].

B. Screening of the cDNA Library

Approximately 2.2×10⁵ primary transformants were divided into 44 poolswith each containing −5000 individual clones. Plasmid DNA was preparedfrom each pool by the CTAB-DNA precipitation method as described [DelSal et al., Biotechniques, 7, 514-519 (1989)]. Two micrograms of eachplasmid DNA pool was digested with restriction enzyme NotI and separatedby gel electrophoresis. Linearized DNA was transferred onto GeneScreenPlus membrane (DuPont) and hybridized with ³²P-labeled PCR generatedhuman SCF cDNA (Example 3) under conditions previously described [Lin etal., Proc. Natl. Acad. Sci. USA, 82, 7580-7584 (1985)]. Three poolscontaining positive signal were identified from the hybridization. Thesepools of colonies were rescreened by the colony-hybridization procedure[Lin et al., Gene 44, 201-209 (1986)] until a single colony was obtainedfrom each pool. The cDNA sizes of these three isolated clones arebetween 5.0 to 5.4 kb. Restriction enzyme digestions and nucleotidesequence determination at the 5′ end indicate that two out of the threeclones are identical (10-1a and 21-7a). They both contain the codingregion and approximately 200 bp of 5′ untranslated region (5′ UTR). Thethird clone (26-1a) is roughly 400 bp shorter at the 5′ end than theother two clones. The sequence of this human SCF cDNA is shown in FIG.42. Of particular note is the hydrophobic transmembrane domain sequencestarting in the region of amino acids 186-190 and ending at amino acid212.

C. Construction of pDSRα2 hSCF-¹⁻²⁴⁸

pDSRα2 hSCF¹⁻²⁴⁸ was generated using plasmids 10-1a (as described inExample 16B) and pGEM3 hSCF¹⁻¹⁶⁴ as follows: The HindIII insert frompGEM3 hSCF¹⁻¹⁶⁴ was transferred to M13mp18. The nucleotides immediatelyupstream of the ATG initiation codon were changed by site directedmutagenesis from tttcctttATG to gccgccgccATG using the antisenseoligonucleotide.

5′-TCT TCT TCA TGG CGG CGG CAA GCT T 3′

and the oligonucleotide-directed in vitro mutagenesis system kit andprotocols from Amersham Corp. to generate M13mp18 hSCF¹⁻¹⁶⁴. This DNAwas digested with HindIII and inserted into pDSRα2 which had beendigested with HindIII. This clone is designated pDSRα2 hSCF¹⁻¹⁶⁴. DNAfrom pDSRα2 hSCF^(K1-164) was digested with XbaI and the DNA made bluntended by the addition of Kenow enzyme and four dNTPs. Followingtermination of this reaction the DNA was further digested with theenzyme SpeI. Clone 10-1a was digested with DraI to generate a blunt end3′ to the open reading frame in the insert and with SpeI which cuts atthe same site within the gene in both pDSRα2 hSCF^(K1-164) and 10-1a.These DNAs were ligated together to generate pDSRα2 hSCF^(K1-248).

D. Transfection and Immunoprecipitation of COS Cells with pDSRα2hSCF¹⁻²⁴⁸ DNA.

COS-7 (ATCC CRL 1651) cells were transfected with DNA constructed asdescribed above. 4×10⁶ cells in 0.8 ml DMEM+5% FBS were electroporatedat 1600 V with either 10 μg pDSRα2 hSCF^(K1-248) DNA or 10 μg pDSRα2vector DNA (vector control). Following electroporation, cells werereplated into two 60-mm dishes. After 24 hrs, the medium was replacedwith fresh complete medium.

72 hrs after transfection, each dish was labelled with ³⁵S-mediumaccording to a modification of the protocol of Yarden et al., (PNAS 87,2569-2573, 1990). Cells were washed once with PBS and then incubatedwith methionine-free, cysteine-free DMEM (met⁻cys⁻DMEM) for 30 min. Themedium was removed and 1 ml met⁻cys⁻DMEM containing 100 μCi/mlTrans³⁵S-Label (ICN) was added to each dish. Cells were incubated at 37°C. for 8 hr. The medium was harvested, clarified by centrifugation toremove cell debris and frozen at −20° C.

Aliquots of labelled conditioned medium of COS/pDSRα2 hSCF^(K1-248) andCOS/pDSRα2 vector control were immunoprecipitated along with mediumsamples of ³⁵S-labelled CHO/pDSRα2 hSCF¹⁻¹⁶⁴ clone 17 cells (see Example5) according to a modification of the protocol of Yarden et al. (EMBO,J. 6, 3341-3351, 1987). One ml of each sample of conditioned medium wastreated with 10 μl of pre-immune rabbit serum (#1379 P.I.). Samples wereincubated for 5 h. at 4° C. One hundred microliters of a 10% suspensionof Staphylococcus aureus (Pansorbin, Calbiochem.) in 0.15 M NaCl, 20 mMTris pH 7.5, 0.2% Triton X-100 was added to each tube. Samples wereincubated for an additional one hour at 4° C. Immune complexes werepelleted by centrifugation at 13,000×g for 5 min. Supernatants weretransferred to new tubes and incubated with 5 μl rabbit polyclonalantiserum (#1381 TB4), purified as in Example 11, against CHO derivedhSCF¹⁻¹⁶² overnight at 4° C. 100 μl Pansorbin was added for 1 h. andimmune complexes were pelleted as before. Pellets were washed 1× withlysis buffer (0.5% Na-dioxycholate, 0.5% NP-40, 50 mM NaCl, 25 mM TrispH 8), 3× with wash buffer (0.5 M NaCl, 20 mM Tris pH 7.5, 0.2% TritonX-100), and 1× with 20 mM Tris pH 7.5. Pellets were resuspended in 50 μl10 mM Tris pH 7.5, 0.1% SDS, 0.1 M β-mercaptoethanol. SCF protein waseluted by boiling for 5 min. Samples were centrifuged at 13,000×g for 5min. and supernatants were recovered.

Treatment with glycosidases was accomplished as follows: threemicroliters of 75 mM CHAPS containing 1.6 mU O-glycanase, 0.5 UN-glycanase, and 0.02 U neuraminidase was added to 25 μl of immunecomplex samples and incubated for 3 hr. at 37° C. An equal volume of2×PAGE sample buffer was added and samples were boiled for 3 min.Digested and undigested samples were electrophoresed on a 15%SDS-polyacrylamide reducing gel overnight at 8 mA. The gel was fixed inmethanol-acetic acid, treated with Enlightening enhancer (NEN) for 30min., dried, and exposed to Kodak XAR-5 film at −70° C.

FIG. 43 shows the autoradiograph of the results. Lanes 1 and 2 aresamples from control COS/pDSRα2 cultures, lanes 3 and 4 fromCOS/pDSRα2hSCF¹⁻²⁴⁸, lanes 5 and 6 from CHO/pDSRα2 hSCF¹⁻¹⁶⁴. Lanes 1,3, and 5 are undigested immune precipitates; lanes 2, 4, and 6 have beendigested with glycanases as described above. The positions of themolecular weight markers are shown on the left. Processing of the SCF inCOS transfected with pDSRα2 hSCF¹⁻²⁴⁸ closely resembles that ofhSCF¹⁻¹⁶⁴ secreted from CHO transfected with pDSRα2 hSCF¹⁻¹⁶⁴, (Example11). This strongly suggests that the natural protelytic processing sitereleasing SCF from the cell is in the vicinity of amino acid 164.

EXAMPLE 17 Quaternary Structure Analysis of Human SCF

Upon calibration of the gel filtration column (ACA 54) described inExample 1 for purification of SCF from BRL cell medium with molecularweight standards, and upon elution of purified SCF from other calibratedgel filtration columns, it is evident that SCF purified from BRL cellmedium behaves with an apparent molecular weight of approximately70,000-90,000 relative to the molecular weight standards. In contrast,the apparent molecular weight by SDS-PAGE is approximately28,000-35,000. While it is recognized that glycosylated proteins maybehave anomalously in such analyses, the results suggest that theBRL-derived rat SCF may exist as non-covalently associated dimer undernon-denaturing conditions. Similar results apply for recombinant SCFforms (e.g. rat and human SCF¹⁻¹⁶⁴ derived from E. coli, rat and humanSCF¹⁻¹⁶² derived from CHO cells) in that the molecular size estimated bygel filtration under non-denaturing conditions is roughly twice thatestimated by gel filtration under denaturing conditions (i.e., presenceof SDS), or by SDS-PAGE, in each particular case. Furthermoresedimentation velocity analysis, which provides an accuratedetermination of molecular weight in solution, gives a value of about36,000 for molecular weight of E. coli-derived recombinant humanSCF¹⁻¹⁶⁴. The value is again approximately twice that seen by SDS-PAGE(˜18,000-19,000). Therefore, while it is recognized that there may bemultiple oligomeric states (including the monomeric state), it appearsthat the dimeric state predominates under some circumstances insolution. CHO cell-derived human/SCF¹⁻¹⁶² has a molecular weight ofabout 53,000 by sedimentation equilibrium analysis; this indicates thatit is dimeric also, and that it is about 30% carbohydrate by weight.

EXAMPLE 18 Isolation of Human SCF cDNA Clones from the 5637 Cell Line

A. Construction of the 5637 cDNA Library

Total RNA was isolated from human bladder carcinoma cell line 5637 (ATCCHTB-9) by the acid guanidinium thiocyanate-phenol-chloroform extractionmethod [Chomczynski et al., Anal. Biochem, 162, 156 (1987)], and poly(A)RNA was recovered by using an oligo(dT) spin column purchased fromClontech. Double-stranded cDNA was prepared from 2 μg poly(A) RNA with aBRL cDNA synthesis kit under the conditions recommended by the supplier.Approximately 80 ng of column fractionated double-stranded cDNA with anaverage size of 2 kb was ligated to 300 ng SalI/NotI digested vectorpSPORT 1 [D'Alessio et al., Focus, 12, 47-50 (1990)] and transformedinto DH5α cells by electroporation [Dower et al., Nucl. Acids Res., 16,6127-6145 (1988)].

B. Screening of the cDNA Library

Approximately 1.5×10⁵ primary transformants were divided into 30 poolswith each containing approximately 5000 individual clones. Plasmid DNAwas prepared from each pool by the CTAB-DNA precipitation method asdescribed [Del Sal et al., Biotechniques, 7, 514-519 (1989)]. Twomicrograms of each plasmid DNA pool was digested with restriction enzymeNotI and separated by gel electrophoresis. Linearized DNA wastransferred to GeneScreen Plus membrane (DuPont) and hybridized with³²P-labeled full length human SCF cDNA isolated from HT1080 cell line(Example 16) under the conditions previously described [Lin et al.,Proc. Natl. Acad. Sci. USA, 82, 7580-7584 (1985)]. Seven poolscontaining positive signal were identified from the hybridization. Thepools of colonies were rescreened with ³²P-labeled PCR generated humanSCF cDNA (Example 3) by the colony hybridization procedure [Lin et al.,Gene, 44, 201-209 (1986)] until a single colony was obtained from fourof the pools. The insert sizes of four isolated clones are approximately5.3 kb. Restriction enzyme digestions and nucleotide sequence analysisof the 5′-ends of the clones indicate that the four clones areidentical. The sequence of this human cDNA is shown in FIG. 44. The cDNAof FIG. 44 codes for a polypeptide in which amino acids 149-177 of thesequences in FIG. 42 are replaced by a single GLy residue.

EXAMPLE 19 SCF Enhancement of Survival After Lethal Irradiation

A. SCF in vivo Activity on Survival After Lethal Irradiation

The effect of SCF on survival of mice after lethal irradiation wastested. Mice used were 10 to 12 week-old female Balb/c. Groups of 5 micewere used in all experiments and the mice were matched for body weightwithin each experiment. Mice were irradiated at 850 rad or 950 rad in asingle dose. Mice were injected with factors alone or factors plusnormal Balb/c bone marrow cells. In the first case, mice were injectedintravenously 24 hrs. after irradiation with rat PEG-SCF¹⁻¹⁶⁴ (20μg/kg), purified from E. coli and modified by the addition ofpolyethylene glycol as in Example 12, or with saline for controlanimals. For the transplant model, mice were injected i.v. with variouscell doses of normal Balb/c bone marrow 4 hours after irradiation.Treatment with rat PEG-SCF¹⁻¹⁶⁴ was performed by adding 200 μg/kg of ratPEG-SCF¹⁻¹⁶⁴ to the cell suspension 1 hour prior to injection and givenas a single i.v. injection of factor plus cells.

After irradiation at 850 rads, mice were injected with rat PEG-SCF¹⁻¹⁶⁴or saline. The results are shown in FIG. 45. Injection of ratPEG-SCF¹⁻¹⁶⁴ significantly enhanced the survival time of mice comparedto control animals (P<0.0001). Mice injected with saline survived anaverage of 7.7 days, while rat PEG-SCF¹⁻¹⁶⁴ treated mice survived anaverage of 9.4 days (FIG. 45). The results presented in FIG. 45represent the compilation of 4 separate experiments with 30 mice in eachtreatment group.

The increased survival of mice treated with rat PEG-SCF¹⁻¹⁶⁴ suggests aneffect of SCF on the bone marrow cells of the irradiated animals.Preliminary studies of the hematological parameters of these animalsshow slight increases in platelet levels compared to control animals at5 days post irradiation, however at 7 days post irradiation the plateletlevels are not significantly different to control animals. Nodifferences in RBC or WBC levels or bone marrow cellularity have beendetected.

B. Survival of Transplanted Mice Treated with SCF

Doses of 10% femur of normal Balb/c bone marrow cells transplanted intomice irradiated at 850 rad can rescue 90% or greater of animals (datanot presented). Therefore a dose of irradiation of 850 rad was used witha transplant dose of 5% femur to study the effects of rat PEG-SCF¹⁻¹⁶⁴on survival. At this cell dose it was expected that a large percentageof mice not receiving SCF would not survive; if rat PEG-SCF¹⁻¹⁶⁴ couldstimulate the transplanted cells there might be an increase in survival.As shown in FIG. 46, approximately 30% of control mice survived past 8days post irradiation. Treatment with rat PEG-SCF¹⁻¹⁶⁴ resulted in adramatic increase of survival with greater than 95% of these micesurviving out to at least 30 days (FIG. 46). The results presented inFIG. 46 represent the compilation of results from 4 separate experimentsrepresenting 20 mice in both the control and rat PEG-SCF¹⁻¹⁶⁴ treatedmice. At higher doses of irradiation, treatment of mice with ratPEG-SCF¹⁻¹⁶⁴ in conjunction with marrow transplant also resulted inincreased survival (FIG. 47). Control mice irradiated at 950 rads andtransplanted with 10% of a femur were dead by day 8, while approximately40% of mice treated with rat PEG-SCF¹⁻¹⁶⁴ survived 20 days or longer.20% of control mice transplanted with 20% of a femur survived past 20days while 80% of rSCF treated animals survived (FIG. 47).

C. Radioprotective Effects of SCF on Lethally Irradiated Mice Without aBone Marrow Transplant

The effects of SCF administration prior to irradiation were compared tothe effects of SCF administration post-irradiation.

Female BDF1 mice (Charles River Laboratories, were used. All mice werebetween 7 and 8 weeks old and averaged 20-24 g each. Irradiationconsisted of a lethal split dose of 575 RADS each (total 1150 RADS)delivered 4 hours apart from a Gamma Cell to 40 duel cobalt source,(Atomic Energy Of Canada Limited).

In the experiment shown in FIG. 19-1, the ability of SCF, administeredprior to irradiation, to save mice from an otherwise lethal exposure wastested. Rat SCF, purified from E. coli as in Example 10 and modified bythe addition of polyethylene glycol as in Example 12, was administeredto two groups of mice (n=30), either intra-peritoneally or intravenouslyat a dose of 100 μg/kg. Control animals received excipient only whichconsisted of phosphate-buffered saline, 0.1% fetal bovine serum. Thetimes of administration were t=−20 hours and t=−2 hours to theirradiation event (t=0). The survival of the animals was monitoreddaily. The results are shown in FIG. 48. Both routes of administrationof rat SCF-PEG enhanced survival of the irradiated mice. At 30 days postirradiation, 100% of the animals treated with SCF were alive, whereasonly 35% of the animals in the control group were alive. Since similarexperiments, outlined in Example 19 A where SCF was administeredpost-irradiation only, yielded different results, the two modes ofadministration were compared directly in a single experiment. Theexperiment was performed as described above for FIG. 49 except thegroups were as follows (irradiation was at t=0): group 1, control; group2, rat SCF-PEG administered at t=−20 hours and t=−2 hours; group 3, ratSCF-PEG administered at t=−20 hours, t=−2 hours, and t=+4 hours; andgroup 4, rat SCF-PEG administered at t=+4 hours only. Both groupsreceiving rat SCF-PEG prior to irradiation survived at 95-100% at day 14(groups 2 and 3). In accordance with the experiment described in Example19 A, the animals receiving rat SCF-PEG post irradiation only did notsurvive the irradiation event, although they survived longer thancontrols.

These experiments demonstrate the utility of SCF administration toprotect against the lethal effects of irradiation. These protectiveeffects are most effective when SCF is administered prior to theirradiation event as well as after. This aspect of in vivo activity ofSCF can be utilized in dose intensification regimes in anti-neoplasticradiotherapy.

EXAMPLE 20 Production of Monoclonal Antibodies Against SCF

8-week old female BALB/c mice (Charles River, Wilmington, Mass.) wereinjected subcutaneously with 20 μg of human SCF¹⁻¹⁶⁴ expressed from E.coli in complete Freund's adjuvant (H37-Ra; Difco Laboratories, Detroit,Mich.). Booster immunizations of 50 μg of the same antigen in IncompleteFreund's adjuvant were subsequently administered on days 14, 38 and 57.Three days after the last injection, 2 mice were sacrificed and theirspleen cells fused with the sp 2/0 myeloma line according to theprocedures described by Nowinski et al., [Virology 93, 111-116 (1979)].

The media used for cell culture of sp 2/0 and hybridoma was Dulbecco'sModified Eagle's Medium (DMEM), (Gibco, Chagrin Falls, Ohio)supplemented with 20% heat inactivated fetal bovine serum (Phibro Chem.,Fort Lee, N.J.), 110 mg/ml sodium pyruvate, 100 U/ml penicillin and 100mcg/ml streptomycin (Gibco). After cell fusion hybrids were selected inHAT medium, the above medium containing 10⁻⁴M hypoxanthine, 4×10⁻⁷Maminopterin and 1.6×10⁻⁵M thymidine, for two weeks, then cultured inmedium containing hypoxanthine and thymidine for two weeks.

Hybridomas were screen as follows: Polystyrene wells (Costar, Cambridge,Mass.) were sensitized with 0.25 μg of human SCF¹⁻¹⁶⁴ (E. coli) in 50 μlof 50 mM bicarbonate buffer pH 9.2 for two hours at room temperature,then overnight at 4° C. Plates were then blocked with 5% BSA in PBS for30 minutes at room temperature, then incubated with hybridoma culturesupernatant for one hour at 37° C. The solution was decanted and thebound antibodies incubated with a 1:500 dilution of Goat-anti-mouse IgGconjugated with Horse Radish Peroxidase (Boehringer MannheimBiochemicals, Indianapolis, Ind.) for one hour at 37° C. The plates werewashed with wash solution (KPL, Gaithersburg, Md.) then developed withmixture of H₂O₂ and ABTS (KPL). Colorimetry was conducted at 405 nm.

Hybridoma cell cultures secreting antibody specific for human SCF¹⁻¹⁶⁴(E. coli) were tested by ELISA, same as hybridoma screen procedures, forcrossreactivities to human SCF¹⁻¹⁶² (CHO). Hybridomas were subcloned bylimiting dilution method. 55 wells of hybridoma supernatant testedstrongly positive to human SCF¹⁻¹⁶⁴ (E. coli); 9 of them crossreacted tohuman SCF¹⁻¹⁶² (CHO).

Several hybridoma cells have been cloned as follows:

Monoclone IgG Isotype Reactivity to human SCF¹⁻¹⁶² (CHO) 4G12-13 IgG1 No6C9A IgG1 No 8H7A IgG1 Yes

Hybridomas 4G12-13 and 8H7A were deposited with the ATCC on Sep. 26,1990.

EXAMPLE 21 Synergistic Effect of SCF and Other Growth Factors

A. Synergistic Effect of SCF and G-CSF in Rodents

Lewis rats, male, weighing approximately 225 gms, were injectedintravenously via the dorsal vein of the penis with eitherpolyethylenesporeglycol-modified ratSCF-PEG (Examples 10 and 12),recombinant human G-CSF, a combination of both growth factors, or withcarrier consisting of 1% normal rat serum in sterile saline.Quantitative peripheral blood and bone marrow differentials wereperformed at various timepoints as previously described [Hulse, ActaHaematol. 31:50 (1964); Chervenick et al., Am. J. Physiol. 215: 353(1968)]. Histologic examination of the spleen was performed withBouin's-fixed paraffin-embedded sections stained withhematoxylin-and-eosin as well as by the Giemsa method. The numbers ofnormoblasts, megakaryocytes, and mast cells per 400X or 1000X high powerfield (HPF) in the spleen was quantitated by counting the number of eachcell type in randomly selected fields of the red pulp. Increases incirculating numbers of neutrophils over extended time periods were whenso stated calculated by planimetry as previously described. [Ulrich etal., Blood 75:48 (1990)]. Data is expressed as the mean plus-or-minusone standard deviation and statistical analysis is by the unpairedt-test.

A single coinjection of ratSCF-PEG (25 ug/rat) plus G-CSF (25 ug/rat)causes an increase in circulating neutrophils that is approximatelyadditive (FIG. 50 CSF) as compared to ratSCF-PEG alone (25 ug/rat) orG-CSF alone (25 ug/rat) as measured by planimetry over a 35 hour timeperiod. The kinetics of ratSCF-PEG plus G-CSF-induced peripheralneutrophilia reflect the combined effect of the differing kinetics ofratSCF-induced neutrophilia peaking at 6 hours and G-CSF-inducedneutrophilia peaking at 12 hours (FIG. 50). The bone marrow at 6 hoursafter a single coinjection of ratSCF-PEG plus G-CSF (Table 18) shows agreater than additive decrease in mature marrow neutrophils(9.94±0.3×10⁶ PMN/humerus in carrier control rats vs. 2.11±0.3×10⁶PMN/humerus in ratSCF-PEG plus G-CSF-treated rats, 79% decrease) ascompared to ratSCF-PEG alone-treated rats (7.55±0.2×10⁶ PMN/humerus, 24%decrease) or G-CSF alone-treated rates (5.55±0.5×10⁶ PMN/humerus, 44%decrease). A significant increase in myeloblasts and promyelocytes wasseen in ratSCF-PEG, G-CSF-, and ratSCF-PEG plus G-CSF-treated rats at 6hours as compared to carrier controls (Table 18), but no significantincrease in any form of immature myeloid cells is noted in ratSCF-PEGplus G-CSF-treated rats as compared to ratSCF-PEG alone- or G-CSFalone-treated rats. A significant increase in myeloblasts is noted at 24hours, however, in the ratSCF-PEG plus G-CSF group as compared to eitherratSCF-PEG, G-CSF, or carrier alone (p<0.01, Table 19).

Daily coinjection of ratSCF-PEG (25 ug/rat) plus G-CSF (25 ug/rat) forone week causes a highly synergistic increase in circulating neutrophils(FIG. 51) as compared to ratSCF-PEG alone (25 ug/rat) or G-CSF alone (25ug/rat). A marked linear increase rise in the number of circulatingneutrophils occurs between day 4 and 6 after the coinjection ofratSCF-PEG plus G-CSF to 41.4±1.2×10³ PMN/mm³ at 24 hours after the lastinjection of the week as compared to 10.6±3.6×10³ PMN/mm³ in G-CSFtreated rats and 2.4±1.3×10³ PMN/mm³ in ratSCF-PEG alone treated rats(FIG. 51). A more detailed kinetic study of ratSCF-PEG plusG-CSF-induced neutrophilia after the last injection of the week showedthat the peak of circulating neutrophils occurs at 12 hours and reachesa level of 69.2±2.5×10³ PMN/mm³ as compared to 25.3±0.3×10³ PMN/mm³ inG-CSF-treated rats and 5.6±3.4×10³ in ratSCF-PEG-treated rats (FIG. 52).The neutrophils of ratSCF-PEG plus G-CSF-treated rats were extremelyhypersegmented (FIG. 52). In addition to the overwhelming increase inmature neutrophils in the circulation, an increase in immature myeloidforms was noted as well as the appearance of immature monocytoid forms,rare macrophage-like cells that contained vacuoles and ingestederythroid or lymphoid cells, rare basophils, rare mononuclearpromegakaryocytic forms and occasional late normoblasts in peripheralblood smears. As many as 3% of the nucleated circulating blood cellswere normoblasts in some of the peripheral blood smears of ratSCF-PEGplus G-CSF-treated rats after daily treatment for one week.

Two of the four rats in the ratSCF-PEG plus G-CSF-treated group died(one on the fifth day and one on the sixth day of the experiment), oneof the surviving rats appeared ill on the day of sacrifice (the seventhday), and both of the surviving rats were thrombocytopenic. None of therats in the ratSCF-PEG alone, G-CSF alone, or carrier control groupsshowed any evidence of morbidity or were thrombocytopenic.

The bone marrow at 24 hours after the daily coninjection of ratSCF-PEGplus G-CSF for one week demonstrated a synergistic increase in matureneutrophils form 10.6±0.6×10⁶ PMN/humerus in carrier controls,14.5±1.0×10⁶ PMN/humerus in ratSCF-PEG alone-treated rats, and28.5±2.1×10⁶ PMN/humerus in G-CSF alone-treated rats (Table 20). Theneutrophils in the marrow are generally hypersegmented and are oftenhypergranulated due to an increase in primary azurophilic granules.

The spleens of ratSCF-PEG plus G-CSF-treated rats were much larger andhistologic examination showed increased myelopoiesis, erythropoiesis,and megakaryocytopoiesis as compared to the spleens of control or singlefactor-treated rats. The spleens of ratSCF-PEG plus G-CSF-treated ratsshowed atrophy of the white pulp concomitant with a tremendous expansionof the red pulp which was replaced by nearly confluent extramedullaryhematopoiesis. The number of granulocytic precursors (myeloblasts tometamyelocytes) was readily seen by scanning histologic sections of thespleen to be markedly increased in the ratSCF-PEG plus G-CSF group ascompared to all other groups. Interestingly, the number of normoblastsin the spleen was also increased in the ratSCF-PEG plus G-CSF group(4.1±5.8 in the ratSCF-PEG alone group, 0±0 in the G-CSF alone group,and 36.4±26.1 in the ratSCF-PEG plus G-CSF group; 18 1,000XHPF/spleen/rat; p<0.0001 comparing ratSCF-PEG plus G-CSF vs. ratSCF-PEGalone). The number of megakaryocytes in the spleen was alsosignificantly increased in the ratSCF-PEG plus G-CSF group (1.8±1.5 inthe ratSCF-PEG alone group, 2.0±1.1 in the G-CSF alone group, and5.2±3.1 in the ratSCF-PEG plus G-CSF group; 12 400X HPF/spleen/rat;p<0.0001 comparing ratSCF-PEG plus G-CSF to either ratSCF-PEG or G-CSFalone).

These results demonstrate that the in vivo combination of ratSCF-PEG andG-CSF causes a synergistic myeloid hyperplasia in the bone marrow andspleen and a synergistic increase in circulating neutrophils. Thesynergism between a single dose of ratSCF-PEG and G-CSF becomes mostdramatically apparent as a rapidly increasing number of circulatingneutrophils between 4 and 6 hours after commencement of administrationof growth factors. Daily coinjection plus G-CSF for one week causes ahighly synergistic increase in circulating neutrophils as compared toratSCF-PEG alone or G-CSF alone.

B. Synergistic Effect of SCF and Other Growth Factors in Canines

Though single factors such as G-CSF have been shown to have importanteffects on hematopoietic recovery, the combination of SCF with G-CSF hasa dramatic hematologic response. In the first set of experiments, 3normal dogs were treated with recombinant canine SCF alone at 200μg/kg/day subcutaneously or by continuous intravenous infusion. Theseanimals responded with an increase in the white blood cell count to30-50,000/mm³, from a baseline of 10-15,000 mm³ by day 8-12. Whenanother group of normal dogs were treated for 28 days with recombinantcanine SCF (200 μg/kg/day SCF and G-CSF (10 μg/kg/day SC), the whiteblood cell count increased from a normal range of 10-11,000/mm³ to200-240,000 cells/mm³ by day 17-21. This demonstrates that the effectsof SCF are dramatically enhanced in combination with other hematopoieticgrowth factors. Similarly, in vitro data show that SCF in combinationwith EPO dramatically enhances BFU-E growth (number and size, seeExample 9), again demonstrating that combinations of hematopoieticgrowth factors are more effective in eliciting a hematopoietic responseand/or may allow for lower doses of other factors to elicit the sameresponse.

EXAMPLE 22 The Use of SCF in Hematopoietic Transplantation

A. The Effects of SCF on Amplification of Bone Marrow and PeripheralBlood Hematopoietic Progenitors

The effects of SCF administration on circulating hematopoieticprogenitors in normal baboons was studied. The experimental design wasidentical to that described in Example 8C. Briefly, normal baboons wereadministered 200 μg/kg/day human SCF¹⁻¹⁶⁴, produced in E. coli as inExample 10 and modified by the addition of polyethylene glycol as inExample 12, as a continuous intravenous infusion. At various times bonemarrow and peripheral blood was harvested and cultured at a density of2×10⁵ per ml in Iscoves' Modified Dulbecco's Medium (Gibco, GrandIsland, N.Y.) in 0.3% (W/v) agar (FMC, Rockland, Me.), supplemented with25% fetal bovine serum (Hyclone, Logan, Utah), and 10⁻⁴2-mercaptoethanol in 35 mm culture dishes (Nunc, Naperville, Ill.).Cells were cultured in the presence of human IL-3, IL-6, G-CSF, GM-CSF,SCF at 100 ng/ml and EPO at 10 U/ml. Cultures were incubated at 37° C.in 5% CO₂ in a humidified incubator. At day 14 of culture, colonies wereenumerated using an inverted microscope. Macroscopic BFU-E were definedas those greater than 0.5 mm in diameter.

Marrow CFU-GM and BFU-E were assayed from four baboons before and at theend of the SCF infusion. The number of colonies per 10⁵ cells, i.e.,CFU-GM (41±12 pre-SCF, 36±post-SCF) and BFU-E (78±28 pre-SCF, 52±26post-SCF), were not statistically different. Given the dramaticincreases in marrow cellularity, the absolute number of CFU-GM and BFU-Ewere estimated to be increased.

A fifth baboon given SCF was studied weekly for changes in peripheralblood and marrow colony-forming cells. In marrow, the incidence ofCFU-GM increased 1.1 to 1.3 fold and BFU-E increased 2.5 to 6.5 fold. Inperipheral blood, however, the incidence of colony-forming cells wasmarkedly increased (25 to 100 fold), and absolute numbers ofcolony-forming cells were increased up to 96 fold for CFU-GM, 934 foldfor BFU-E, and greater than 1000 fold for the most primitivecolony-forming cells, CFU-MIX. This expansion of colony-forming cellswas apparent after as little as seven days of SCF administration and wasmaintained throughout the period that SCF was given.

B. Use of SCF in Bone Marrow Transplantation

As noted above, there are several ways that SCF is useful to improvehematopoietic transplantation. One method, as illustrated above is touse SCF to augment the harvest of bone marrow and/or peripheral bloodprogenitors and stem cells by pretreating the donor with SCF. Anotheruse is to treat the recipient of the transplanted cells with SCF afterthe patient has been infused. The recipient is treated with SCF alone orin combination with other early and late acting recombinanthematopoietic growth factors, including EPO, G-CSF, GM-CSF, M-CSF, IL-1,IL-3, IL-6, etc.

SCF alone enhances hematopoietic recovery following bone marrowtransplantation. A variety of experimental variables have been tested ina canine model of bone marrow transplantation, Schuening et al., 76636-640. In one set of experiments for the present invention, dogsreceived either G-CSF or SCF after 920 cGy of total body irradiation and4×10⁸ mononuclear marrow cells per kilogram from a DLA-identicallittermate. The hematologic recovery, as measured by day of neutrophilrecovery to 500 or 1000/mm³, is accelerated when either SCF or G-CSF isadministered compared to control animals that received no growth factor(Table 21). Recovery was 2-6 days earlier in animals that received SCFthan it was in those that received no growth factor. As noted above,combinations of appropriate growth factors with SCF will accelerate andenhance the response to those growth factors following hematopoietictransplantation.

TABLE 21 Effects of rcG-CSF and SCF on Recovery From DLA-indenticalLittermate Marrow Transplantation¹ Recovery of Recovery of TreatmentANC > 500 mm³ ANC > 1000/mm³ Control Day 10 Day 14 rcG-CSF² Day 7 Day 8rcSCF³ #1 Day 7 Day 8 rcSCF³ #2 Day 8 Day 9 ¹920 cGY TBI followed byinfusion of 4 × 10⁸ mononuclear cells per kg DLA-identical lettermatebone marrow ²rcG-CSF administered 10 μg/kg/day_(SC) for 10 daysfollowing transplant ³rcSCF administered 200 μg/kg/day_(SC) for 10 daysfollowing transplant

This aspect of SCF in vivo biological activity can be utilized toenhance the recovery from marrow ablative therapy if the peripheralblood or bone marrow is harvested after SCF administration and thenreinfused after the ablative regimen (i.e., in bone marrowtransplantation or peripheral blood autologous transplantation).

EXAMPLE 23 Effect of SCF on Platelet Formation

Balb/c mice (female, 6-12 weeks of age, Charles River) were treated withrratSCF-PEG (100 ug/kg/day) or excipient control, subcutaneously, 1 timedaily for 7 days (n=7). Blood was sampled through a small incision inthe lateral tail vein on the indicated days after cessation of SCFtreatment. Twenty microliters blood were collected directly into 20 ulmicrocapillary tubes and immediately dispensed into the manufacturersdiluent for the Sysmex Cell Analyzer. Data points are the mean of thedata, error bars are standard error of the mean. Blood platelet countswere determined at the time points indicated in FIG. 53. Platelet countsrose to approximately 160% of control values by Day 4 post-SCF, fell tonormal by Day 10, and rose again to 160% of normal by Day 15. Plateletcounts stabilized at control values by Day 20.

A dose response curve of the SCF effect on platelet counts was generatedwhen Balb/c mice were treated as above with 10, 50, or 100 ug/kg/dayrratSCF-PEG (n=7). Blood was collected and analyzed on the fourth dayfollowing cessation of SCF treatment. These data are shown in FIG. 54and demonstrate that concentrations of rratSCF-PEG between 50-100ug/kg/day are optimal in inducing a rise in platelet counts. Recombinantrat SCF-PEG administration to normal mice also resulted in an increasein platelet size and in the number of megakaryocytes found in the spleenand bone marrow (Table 22). Roden megakaryocytes were identified byexpression of the enzyme acetylcholinesterase (ACH+) which was detectedby cytochemical assays, [Long, Blood 58:1032 (1981)].

Certain similarities were noted between mice given SCF and mice duringrebound thrombocytosis after experimental induction of thrombocytopenia.FIG. 55 demonstrates one model of experimental thrombocytopenia, namelythat of treatment of 5-fluorouracil (5-FU). Balb/c mice were eitheruntreated or treated intravenously with 5-fluorouracil (150 mg/kg) onDay 0 (n=5). Blood analyses were performed on the indicated days as inlegend to FIG. 53. Error bars are present, but not discernable, in someof the control points. As has been demonstrated in the past [Radley etal., Blood 55:164 (1980)], animals become thrombocytopenic by Day 5post-5-FU. However, by Day 12 animals were in a state of reboundthrombocytosis where platelet counts far exceed normal (the “overshoot”effect). After Day 12, platelet counts appeared to cycle from normal tohigh levels throughout the 40 day testing period. As shown in FIG. 56,megakaryocyte number also rise dramatically after 5-FU appearing firstin the bone marrow (Panel A) and then in the spleen (Panel B). Themegakaryocyte numbers were determined in parallel with that shown inFIG. 55. Two Balb/c mice per group were sacrificed at the indicateddays. Cells from bone marrow (Panel A) or spleen (Panel B) werealiquoted at 100,000/well of a microtiter plate and stained foracetylcholinesterase according to published procedures, Long et al.,Immature megakaryocytes in the mouse: Morphology and quantitation byacetlycholinesterase staining. Blood 58: 1032, 1981. Data points are thepercentage of ACH+ cells per well for individual animals.

Platelet volumes also increase after 5-FU (FIG. 57). The data in thisfigure were generated from the same blood samples collected in FIG. 55.Mean Platelet Volume (MPV) is one of the parameters analyzed by theSysmex Cell Analyzer.

The possibility of a relationship between SCF and the physiologicalregulator of platelet production induced in the 5-FU thrombocytopenicmodel was explored. 5-FU was given to normal mice and SCF mRNAexpression levels quantitated in bone marrow cells collected on the daysindicated in FIG. 58. In FIG. 58, one million cells were lysed in SDSbuffer and the lysate was analyzed for the presence of mRNA specific formurine SCF. Probes for mouse SCF or human actin mRNA (which detects thecorresponding murine mRNA) were generated by runoff transcription ofcloned gene regions in vectors containing SP6 or T7 promoters using³⁵S-UTP according to standard protocols (Promega Biotech), or fromsynthetic oligonucleotide partial duplexes, Mulligan et al., Nuc. AcidsRes. 15:8783 (1987). RNA sense strand standards for quantitation of thehybridization assays were produced by runoff transcription of the sameregion in the direction opposite to the direction of probe synthesisusing tracer quantities of ³⁵S-UTP and 0.2 mM unlabeled UTP.

SCF or actin mRNA levels were quantitated as follows. Bone marrow cellswere explanted from animals at the given time post-5FU, enriched forlight density cells by centrifugation on 65% Percoll (Pharmacia;Pistcataway, N.J.) and lysed at 3×10⁶ nucleated cells/ml in 0.2% SDS, 10mM Tris pH 8, 1 mM EDTA, 20 mM dithiothreitol and 100 ug/ml proteinase K(Boerhinger Mannheim; Indianapolis, Ind.). Samples (30 ul) were added to70 ul of hybridization mix consisting of 30 ug/ml yeast tRNA, 30 ug/mlcarrier DNA, 145,000 CPM/ml ³⁵S-labeled probe in 3.0-3.7 M sodiumphosphate, pH 7.2 (depending on length of probe). Samples were incubatedat 84° C. for 2-3 hours then cooled to room temperature before additionof RNase A to 0.03 mg/ml and RNase T1 to 5000 U/ml. Samples wereincubated at 37° C. for 20 minutes before addition of 120 ul of 0.0025%bromophenol blue in formamide. Entire sample was then loaded onto 3.8 mlSephacryl S200 Superfine gel filtration column (0.7 cm×10 cm) and elutedwith 2.0 mls of 10 mM Tris pH 8, 1 mM EDTA, 50 mM NaCl. Effluentscontaining hybridized RNA duplexes were collected directly intoscintillation vials. After addition of 5 mls Liquiscint (New EnglandNuclear; Boston, Mass.) samples were counted 20 minutes or to 3% error.CPM were converted to molecules mRNA by comparison to the linear portionof the standard curve (correlation coefficient=0.97). The data point foreach sample is the mean of replicate tests; bone marrow samples from 3individual animals were taken for each time point so that the data shownis the mean of those determinations. Error bars are standard error ofthe mean. Statistical significance is assigned as described above.

SCF mRNA levels rose dramatically at Days 5 and 7, coinciding exactlywith the nadir of platelet counts immediately preceding thrombocytosis(FIG. 58).

The data in this section show that SCF is active as a thrombopoieticagent in vivo and furthermore that SCF may be involved in thephysiological regulation of platelet production after 5-FU-inducedthrombocytopenia.

Table 22. Megakaryocyte and platelet parameters measured on fourth dayfollowing SCF administration in vivo.

% Ach + % Ach + Platelet Cells in Cells in Factor Count MPV* SpleenMarrow none 1018 +/− 29 6.07 +/− 0.5 .22 +/− .3 .02 +/− .01 SCF** 1429+/− 56 6.24 +/− .05 .85 +/− .9 .59 +/− .05 *MPV; mean platelet volume**ratSCF-PEG administered SC 2 × daily for 7 days at 100 μg/kg/day. Datawere collected 4 days later after last injection.

EXAMPLE 24 Treatment of Bone Marrow Failure States

A variety of congenital and acquired disorders of hematopoiesis havebeen reported to cause clinically significant reductions in the numberof mature circulating peripheral blood cells of one or more lineages.Therefore, the existing data supports that these disorders are treatablewith SCF. For example, aplastic anemia is a clinical syndromecharacterized by pancytopenia due to reduced or absent production ofblood cells in the bone marrow. It is heterogeneous in severity,etiology and pathogenesis.

Most attention has focused on abnormalities of the hematopoietic stemcell, microenvironment or immunologic injury of one of these. Theresponse to immunosuppressive therapy is variable and incomplete.Because aplastic anemia is a defect of the hematopoietic stem cell orproliferative signals from the microenvironment, and is modeled by theSteel mouse [Zsebo et al., Cell 63 213 (1990)], this disorder issuccessfully treated with SCF.

Another bone marrow failure disorder which is responsive to SCF isDiamond-Blackfan anemia (DBA) or congenital pure red cell aplasia. Thiscongenital abnormality results in a selective defect in the productionof red blood cells and often results in chronic transfusion dependency.In vitro data indicate that the defect is overcome by the addition ofexogenous SCF. Bone marrow from patients with DBA (or control marrow)was cultured with or without SCF (100 ng/ml) in the presence oferythropoietin (EPO) (1-5 U/ml), EPO plus IL-3 (1-1000 U/ml), EPO plusGM-CSF (>100 U/ml), or EPO plus lymphocyte-conditioned media (2-5%).Culture of bone marrow from patients with DBA demonstrate two patternsof response to SCF. The majority were hyper-responsive to SCF and showedapproximately 3 fold increase in the frequency of BFU-E at less than orequal to 10 ng/ml, as well as an increase in the size of BFU-E atconcentrations up to 200 ng/ml. Control marrow demonstrated only a 1.5fold increase in frequency of BFU-E. This pattern of response to SCFcould indicate a defect in endogenous SCF and/or its production by themicroenvironment in this group of patients with DBA. The other group ofpatients with DBA demonstrated an increase in the frequency of BFU-E atconcentrations of SCF greater than or equal to 50 ng/ml. This pattern ofresponse reflects an intrinsic defect in the receptor for SCF (c-kit) onthe progenitor cell. In either case (abnormal production of SCF by themicroenvironment or decreased stimulation of the hematopoiesis whichcharacterizes bone marrow failure syndromes such as DBA.

Other bone marrow failure syndromes that are treatable with SCF include,but are not limited to: Fanconi's anemia, dyskeratosis congenita,amegakaryocytic thrombocytopenia, thrombocytopenia with absent radii,and congenital agranulocytosis (e.g. Kostmann's syndrome,Shwachman-Diamond syndrome) as well as other causes of severeneutropenia such as idiopathic and cyclic neutropenia. Severe chronicneutropenia congenital, cyclic or idiopathic are treatable withrecombinant G-CSF.

Cyclic neutropenia, in particular, is a defect in the regulation of stemcell division since other lineages (e.g., platelet, erythrocyte andmonocyte) are also effected. In the canine model of cyclic neutropenia,the cycling of neutrophils, as well as other lineages, is sharplyreduced or even eliminated by SCF treatment. A typical dog with cyclicneutropenia was treated with rcanineSCF (recombinant canine SCF) at 100mg/kg/day subcutaneously over several weeks. The typical 21 day cyclefor neutrophils was eliminated during the first predicted cycle and thesecond predicted nadir was significantly atenuated. This is in contrastto treatment with G-CSF which increases the frequency and amplitude ofneutrophil cycling, but does not eliminate it. Thus, SCF is useful intreating a variety of bone marrow failure syndromes, either alone or incombination with other hematopoietic growth factors.

EXAMPLE 25 SCF Treatment of Patients With HIV-1 Infection

A. Source and Preparation of Peripheral Blood Mononuclear Cells

Leukopaks were obtained from HIV-, CMV-, and EBV-seronegative normaldonors from the American Red Cross. Peripheral blood was obtained from 6patients with HIV-infection after informed consent was obtained. Twopatients were asymptomatic, one has AIDS-related complex and three hadAIDS. None of the 6 patients had received zidovudine within the last sixmonths. None of the patients were anemic (hemoglobin<135 g/L) at thetime of study. All studies were conducted in accordance with UCLA HumanSubject Protection Committee regulations.

Peripheral blood mononuclear cells were isolated from leukopaks andperipheral blood using ficoll-hypaque sedimentation followed byextensive washing with Hank's Balance Salt Solution (HBSS). Blood cellswere enumerated and viability ascertained by trypan blue dye exclusion.

B. Burst Forming Unit Erythro (BFU-E) Assay

Assays for BFU-E were performed in a standard protocol using normalhuman bone marrow as the control. Heparinized blood was diluted with anequal volume of HBSS (GIBCO, Grand Island, N.Y.), layered overFicol-Paque (Pharmacia, Piscataway, N.J.) and centrifuged at 400 g for30 minutes at room temperature. Light density cells (s.g. <1.077) werecollected and washed twice in HBSS. Cells were resuspended in Iscove'sMedium with 10% Fetal Bovine Serum (GIBCO, Grand Island, N.Y.) at aconcentration of 1×10⁷/ml. Cells (1×10⁵) were cultured in Iscove's Mediasupplemented with 5×10⁻⁵ M 2-Mercaptoethanol (2ME) (Sigma Chemicals, St.Louis, Mo.), 30% Fetal Bovine Serum (GIBCO Grand Island, N.Y.), andeither 1 or 4 units of human recombinant erythropoietin (Amgen Inc.,Thousand Oaks, Calif.) in 0.3% agar. Four concentrations of E. coliderived human stem cell factor (hSCF¹⁻¹⁶⁴), obtained as described inExamples 6 and 10, were added (0, 10, 100 and 1000 ng/ml). Zidovudine(AZT) was added to the mixture resulting in final concentrations of 0,0.01 μM, 0.1 μM, 1.0 μM. Erythroid burst colonies were scored after 14days of culture in a humidified atmosphere containing 5% CO₂. Each assaywas done in duplicate and colonies with >50 cells present on day 14 withhemoglobinization were scored as BFU-E.

The 50% inhibitory concentration for zidovudine was calculated byexpressing the mean of four determinations of BFU-E for each level ofzidovudine and huSCF as a percentage of control (no added zidovudine).Linear regression was used to calculate the slope of inhibition. The 50%inhibitory concentration was calculated by interpolation and the valueused as the exponent for the base of 10. This results in directcalculation of the ID₅₀. The r² for all the slopes were >0.90.

C. Effects of HuSCF on Stimulated Peripheral Blood Mononuclear Cells

Peripheral blood mononuclear cells were isolated from the leukopaks oftwo additional normal donors as described above. Cells were resuspendedin Iscove's Modified Dulbecco's Medium containing 20% fetal bovineserum, penn/strep, 1.0% (Sigma Chemical, St. Louis, Mo.) and 10 units/mlof interleukin-2 (Amgen Inc. Thousand Oaks, Calif.). Four concentrationsof human stem cell factor (0, 10, 100, 1,000 ng/ml) were added to themedia. Complete lymphocyte subset analysis of cellular antigens wereanalyzed in duplicate by two color fluorescent cytometry on day 0, 3, 7and 10. Differences in percentages of cell populations were detectedusing independent and paired t-tests (2-tailed). Comparisons were madebetween drug-treated and non-drug-treated values for a single day andbetween single days values and baseline. Cytometric analysis was done induplicate.

D. Results

Exposure of peripheral blood mononuclear cells to erythropoietin andhuman stem cell factor (HuSCF) resulted in a dose-dependent increase inBFU-E formation in the 2 normal patients studied (FIG. 59A). Significantincreases (up to 100%) were seen with concentrations of human stem cellfactor between 10 and 1,000 ng/ml. Near maximal activity was seen at 10ng/ml suggesting that lower concentrations may be active. There weresignificant increases in BFU-E when the dose of erythropoietin wasincreased from 1 IU to 4 IU/ml (FIG. 59B). The colonies observed weresignificantly larger in size than the bursts seen in the absence ofHuSCF.

In the 6 HIV-infected individuals studied, significant dose-dependentincreases in BFU-E were also seen with HuSCF treatment (FIG. 60).Although the number of BFU-E in the absence of HuSCF was markedlyreduced compared to normal (range 2-26 BFU-E/10⁵ peripheral bloodmononuclear cells compared to approximately 74 BFU-E/10⁵ PBMC fornormals), the percentage increases in BFU-E were significantly higher inthe HIV-infected individuals. Near normal numbers of BFU-E were obtainedfor 2 individuals at the 1,000 ng/ml concentration of HuSCF. Althoughthe absolute number of BFU-E seen for some of the patients were stillwell below normal, all 6 individuals responded in vitro to HuSCF.

Because previous studies showed that cytokines could alter theintracellular uptake or intracellular metabolism of deoxynucleosides.[Perno et al., J. Exp. Med. 169:933(1989)] the capacity of hSCF tomodulate the inhibition of red cell progenitors by zidovudine wasevaluated. Each of the normals and all of the HIV individuals had BFU-Eassays performed in the presence and absence of 3 concentrations ofzidovudine and 4 concentrations of huSCF. As observed, (FIGS. 59 andsize of BFU-E bursts) the addition of HuSCF markedly reduced inhibitionof early red cell progenitors by zidovudine. Significant alterations inthe 50% inhibitory dose of zidovudine for BFU-E was seen at all threeconcentrations of human stem cell factor. The IC₅₀ (fifty percentinhibitory concentration) ranged from 2.65 to 1376 μM of zidovudine(FIG. 61). All three of these inhibitory concentrations of zidovudineare well above normal serum levels obtained after 1,000 mg/day ofzidovudine [Klecher et al., Clin. Pharmacol. Ther.; 41:407-12 (1987)].Similar results were observed for all 6 individuals infected with HIV.However, because of the few number of red cell progenitors in 2 of thepatients, the increases in the 50% inhibitory concentrations ofzidovudine for BFU-E did not reach statistical significance.Nonetheless, the trends were clearly present and replicated the effectsof human stem cell factor on BFU-E in the presence of zidovudine in thenormal individuals.

The effect of SCF on the protection of bone marrow derived cells as wellas peripheral blood progenitors (above) was examined. Normal human bonemarrow was prepared as described above for peripheral blood progenitors.Bone marrow cells were exposed to different concentrations of AZT(zidouvidine), and the protective effects of SCF for both erythroid aswell as myeloid cells was determined in semi-solid cultures. Colonieswere scored after 14 day incubation as described above. The results forthe protection of bone marrow derived erythroid cells (FIG. 62) andmyeloid cells (FIG. 63) are indicated. As is seen for peripheral blood,SCF protects bone marrow cells from AZT as well. Another toxic compoundused to fight the opportunistic infections associated with HIV infectionis ganciclovir. Once again, SCF protects bone marrow cells against thetoxic effects of ganciclovir for both erythroid development (FIG. 64)and myeloid development (FIG. 65).

In summary, this example details the effects of HuSCF on early red bloodcell progenitors. Exposure to HuSCF in vitro resulted in a dose andtime-dependent increase in red blood cell progenitors and significantlyaltered the inhibition of red cell progenitors by zidovudine. This wasobserved in both normal and HIV-infected study populations. HuSCF had noeffect on HIV virus replication in primary monocytes or primary humanlymphocytes not did it alter the efficacy of 2′,3′,-dideoxynucleosideanalogues. This is a significant difference from other cytokines whichhave effects on red cell progenitors such as granulocyte-macrophagecolony-stimulating factor (GM-CSF) and interleukin-3 (IL-3). As shown inother studies [Koyanagi et al., Science 241:1773 (1981); Folks et al.,Science 238:800 (1987); Hammer et al., Proc. Natl. Acad. Sci. USA83:8734 (1986)], both GM-CSF and IL-3 significantly increase replicationof HIV in partially purified primary peripheral blood monocytes.

These studies demonstrate that human stem cell factor (HuSCF) is anideal candidate drug for use as adjunctive therapy in the treatment ofHIV-related pancytopenia. This cytokine appears to directly stimulatehuman hematopoietic progenitor cells and synergizes with IL-7, G-CSF,GM-CSF, and IL-3 in the production of pre-B lymphocytes, megakaryocytes,monocytes, granulocytes, and mast cells [Martin et al., Cell 63:203-211(1990); Zsebo et al., Cell, 63:213-224 (1990)].

EXAMPLE 26 Use of Stem Cell Factor to Facilitate Gene Transfer intoHematopoietic Stem Cells

The in vitro survival and proliferation of primitive stem cells iscritical to the success of gene transfer mediated by retroviralinsertion or other known methods of gene transfer. The effect of SCF onthe in vitro maintenance and/or proliferation of primitive progenitorcells has been studied in two systems which have been describedpreviously [Bodine et al., Proc. Natl. Acad. Sci. 86 8897-8901, 1989].The first is a pre-CFU-S assay wherein bone marrow cells are incubatedfor up to six days in suspension culture in the presence of growthfactors. Aliquots are injected into lethally iradiated mice and the micesacrificed at 12-14 days for quantitation of spleen focus formation.IL-3 and IL-6 synergize in enhancing the proliferation of CFU-S between2-6 days in culture. The second is a competitive repopulation assaywhich measures the effects of growth factors on recovery and biologicalactivity of cells capable of sustaining long-term hematopoiesis. Cellsfrom two congenic strains of mice differing for a hemoglobin marker areincubated in suspension independently, cells from one strain as acontrol and cells from a second under experimental conditions. Afterincubation, equal numbers of bone marrow cells from both cultures aremixed and injected into W/W^(v) recipients.

Rat SCF has been evaluated both in the pre-CFU-S and competitiverepopulation assays. SCF alone has very little activity in the pre-CFU-Sassay, similar to IL-3 alone. For enhancing CFU-S activity, thecombination of SCF and IL-3 is equivalent to the previous optimalcombination of IL-3 and IL-6 whereas the combination of SCF and IL-6 is5-fold more active than IL-3 and IL-6 (FIG. 66). A most advantageouscombination is SCF, IL-3 and IL-6; it is 6-fold more active than thecombination of IL-3 and IL-6.

In the competitive repopulation assay, the repopulating ability of cellscultured in the combination of SCF and IL-6 is superior at 35 days(short-term reconstitution) (FIG. 67). A most advantageous combinationfor long term reconstitution is SCF, IL-3 and IL-6, approximately1.5-fold greater than any combination of two factors. Based on thesedata, a most advantageous combination of soluble growth factors forenhancing retroviral mediated gene transfer into stem cells would beSCF, IL-3 and IL-6.

SCF presentation by stromal cells induces the proliferation of primitivebone marrow progenitors. The ultimate in vitro stimulus forproliferation of stem cells is provided by stromal cell linestransfected with human SCF cDNAs with sequences as shown in FIGS. 42 and44. When human bone marrow is cultured on artificial feeder layersexpressing the membrane bound form of human SCF 220 (FIG. 44), there isa continued proliferation of hematopoietic progenitors over time. Anexample of this is given in Table 23. Stromal cells derived from S1/S1embryos prior to their death in utero [Zsebo et al., Cell 63 213 (1990)]were transfected with human SCR cDNAs (either expressing the 220, FIG.44 or 248, FIG. 42, amino acid forms of SCF] and used as feeder layersfor human marrow. Briefly, adherent layers were treated with mitomycin Cand plated at confluence in 6 well plates. Normal human bone marrow,7.5×10⁵ adherence depleted cells, were plated in 5 ml of Iscove'sModified Dulbeccos Medium (Gibco), 10% fetal calf serum, and 10-6 Mhydrocortisone onto the transfected adherent layers. At the indicatedtime points, cells were withdrawn and plated in semi-solid agar usingEPO and IL-3 as a stimulus. For the experiment in Table 24, normaladherence depleted human bone marrow was first enriched forhematopoietic progenitors expressing the CD34 antigen using magneticparticle concentration [Dynal, Inc., Great Neck, N.Y.] prior to platingon the adherent feeder cells. In this case, 3.5×10⁴ cells were culturedon top of the adherent layers as described above. At the indicated timepoints, cells were withdrawn from the cultures and plated in semi-solidagar as described above. For both experiments, colony formation wasenumerated after 14 days of culture in a humidified atmosphere. Thegeneration of colony forming cells over time was enumerated. As isindicated, the membrane bound form of SCF (220 amino acid, FIG. 44) ismore potent at supporting hematopoiesis over time.

The S1/S1 cell line expressing human SCF¹⁻²²⁰ amino acid form isadvantageous for retroviral mediated gene transfer into hematopoieticstem cells. Human bone marrow is infected with retrovirus in thepresence of mammalian cells expressing human SCF¹⁻²²⁰. In addition, theviral producer line optimally is transfected with the human SCF¹⁻²²⁰gene and used for the viral infection as a co-culture.

TABLE 23 Generation of colony forming cells from normal human bonemarrow by cells expressing different splice variants of human SCF. Daysof Cells; Culture CFU-Macs CFU-GM BFU-E CFU-Mix S1/S1-4 7 1.3+/−1  6+/−3 3+/−3 0 14 0 0 0 0 21 0 0 0 0 S1/S1-4 7 31+/−13 51+/−8  3+/−2 0SCF 220 14 57+/−2  69+/−5  0 0 21 46+/−16 23+/−13 0 0 S1/S1-4 7 57+/−1489+/−7  11+/−8  1+/−1 SCF 248 14 5+/−4 9+/−5 5+/−3 0 21 1+/−1 0 0 0

TABLE 24 Generation of colony forming cells from CD34+ bone marrow cellsexpressing different splice variants of human SCF. Days of Totalcolonies/culture well Cells; Culture CFU-Macs CFU-GM BFU-E CFU-MixS1/S1-4 7 4+/−2 10+/−6  11+/−3   1+/−1 14 0 0 0 0 21 0 0 0 0 S1/S1-4 790+/−7  70+/−2  18+/−10 13+/−4 SCF 220 14 14+/−13 60+/−11 2+/−1 0 2136+/−3  23+/−5  0 0 S1/S1-4 7 260+/−64  135+/−20  80+/−20 15+/−5 SCF 24814 0 0 0 0 0 0 0

EXAMPLE 27 Further Characterization of Recombinant Human SCF Obtainedfrom E. coli or CHO Cells

As noted in Example 10, human [Met⁻¹]SCF¹⁻¹⁶⁴ from E. coli has aminoacid composition and amino sequence expected from analysis of the gene.Using the methods outlined in Example 2, it has been determined thathuman SCF¹⁻¹⁶⁵ obtained from E. coli as described in Example 10 also hasthe amino acid composition and amino acid sequence expected fromanalysis of the gene, and also retains Met at position (−1).

Purified E. coli-derived human [Met⁻¹]SCF¹⁻¹⁶⁴ and CHO cell-derivedhuman [Met⁻¹]SCF¹⁻¹⁶² have been studied by methods indicative ofsecondary and tertiary structure. Fluorescence emission spectra, withexcitation at 280 nm, have been obtained. These are shown in FIG. 68.The molecules were dissolved in phosphate-buffered saline. The spectraconsist of a single peak with a maximum at 325 nm, and a full width athalf maximum (FWHM) of between 45 and 50 nm. Both the emissionwavelength and the FWHM suggest that the single Trp is present in ahydrophobic environment, and that this environment is the same in bothmolecules.

Circular dichroism studies have also been carried out. FIG. 69 shows thefar ultraviolet (UV) spectra and near UV spectra (B) for the E.coli-derived SCR (solid lines) and CHO cell-derived SCF (dotted lines).The molecules were dissolved in phosphate-buffered saline. The far UVspectra contain minima at 208 nm and 222 nm. Using the Greenfield-Fasmanequation [Greenfield and Fasman, Biochemistry 8, 4108-4116 (1969)], thespectra suggest 47% α-helix, while the method of Chang et al. [Anal.Biochem. 91, 13-31 (1978)] indicates about 38% α-helix, 33% β-sheet, and29% disordered structure. The near UV spectra have minima at 295 nm and286 nm attributable to tryptophan, minima at 261 nm and 268 nmattributable to phenylalanine, and minima at 278 probably attributableto tyrosine, with some overlap between chromophores. The resultsindicate that the aromatic chromophores are located in asymmetricenvironments. Both the far UV and near UV results are the same for E.coli-derived SCF and CHO cell-derived SCF, indicating similarity ofstructure.

Second derivative infrared spectra in the amide I region (1700-1620cm⁻¹) of the E. coli-derived SCF (A) and CHO cell-derived SCF (B) areshown in FIG. 70. These spectra are related to polypeptide backboneconformation [Byler and Susi, Biopolymers 25, 469-487 (1986); Surewiczand Mantsch, Biochim. Biophys. Acta 952, 115-130 (1988)] and areessentially identical for the two proteins. Band assignments [Byler andSusi (1986), supra; Surewicz and Mantsch (1988), supra] allow one toestimate that the two SCFs have predominantly helical structures, ˜31%α-helix and 19% 3₁₀-helix, with lesser fractions of β-strands (˜25%),turns (˜15%), and disordered structures (˜14%).

Disulfide structure of various molecules referred to in previousexamples have been determined. These include BRL 3A cell-derived naturalrat SCF, E. coli-derived rat [Met⁻¹]SCF¹⁻¹⁶⁴, CHO cell-derived ratSCF¹⁻¹⁶² , E. coli-derived human [Met⁻¹]SCF¹⁻¹⁶⁴ , E. coli-derived human[Met⁻¹]SCF¹⁻¹⁶⁵, and CHO cell-derived human SCF¹⁻¹⁶². The methods usedinclude those outlined in Example 2 for amino acid sequence andstructure determination. The proteins are digested with proteases, andthe resulting peptides isolated by reverse-phase HPLC. If this is donewith and without prior reduction, it is possible to isolate and identifydisulfide-linked peptides. Isolated disulfide-linked peptides can alsobe identified by plasma desorption mass spectroscopy. By such methods ithas been demonstrated that all of the above-mentioned molecules haveintrachain disulfide bonds linking Cys-4 and Cys-89, and linking Cys-43and Cys-138.

EXAMPLE 28 Production and Characteristics of SCF Analogs and FragmentsExpressed in E. coli

Plasmid constructions for expression of numerous SCF analogs andfragments have been made. Site-directed mutagenesis has been used toprepare plasmids with initiating methionine codon followed by codons foramino acids 1 to 178, 173, 168, 166, 163, 162, 161, 160, 159, 158, 157,156, 148, 145, 141, and 137, using the numbering of FIG. 15C. The DNAfor human SCF¹⁻¹⁸³ (Example 6B) was cloned into MP11 from Xba1 to BamH1.Phage from this cloning was used to transfect an E. coli dut⁻ ung⁻strain, R21032. Single stranded M13 DNA was prepared from this strainand site-directed mutagenesis was performed (reference IL-2 patent).After the site-directed mutagenesis reactions, the DNAs were transformedinto an E. coli dut⁺ ung⁺ strain, JM101. Clones were screened andsequenced as described in copending U.S. patent application Ser. No.717,334, filed Mar. 29, 1985. Plasmid DNA preps were made from positiveclones and the SCF regions from Xba1 to BamH1 were cloned into pCFM1656as described in copending U.S. patent application Ser. No. 501,904,filed Mar. 29, 1990. The oligonucleotides for each cloning were designedto substitute a stop codon for an amino acid codon at the appropriateposition for each analog.

Plasmids with initiating methionine codon followed by codons for aminoacids 1 to 130, 120, 110, 100, 133, 127, and 123 (using the numbering ofFIG. 42) have been made using the polymerase chain reaction. ThepCFM1156 human SCF¹⁻¹⁶⁴ plasmid DNA (Example 6B) was used to prime thereaction using a 5′ oligonucleotide 5′ to the Xba1 site and a 3′oligonucleotide which included a direct match to the desired 3′ end ofthe analog DNA, followed by a stop codon, followed by a BamH1 site.After the polymerase chain reaction, the polymerase chain reactionfragments were cleaved with Xba1 and BamH1, gel purified, and clonedinto pCFM1656 cut with Xba1 and BamH1.

Plasmids with initiating methionine codon followed by codons for aminoacids 2 to 164, 5 to 164, and 11 to 164 (using the numbering of FIG. 42)were also made using polymerase chain reaction. The pCFM1156 humanSCF¹⁻¹⁶⁴ plasmid DNA (Example 6B) was used with two primers. The 5′oligonucleotide primer included an Nde1 site (which includes the ATGcodon for the initiating methionine) and a homologous stretch of DNAstarting at the codon for the first desired amino acids. The 3′oligonucleotide primer was totally homologous and was 3′ to the EcoR1site in the gene. After the polymerase chain reaction, the fragment wascut with Nde1 and EcoR1, gel purified, and cloned back into the pCFM1156human SCF¹⁻¹⁶⁴ plasmid cut with Nde1 and EcoR1.

A plasmid with initiating methionine codon followed by codons for aminoacids 1 to 248 (using the numbering of FIG. 42) was made using DNAobtained directly from the cDNA clone (Example 16). The cDNA was cleavedwith Spe1 and Dra1 (blunt end) and the fragment with the SCF region wasgel purified. This was cloned into the pCFM1156 human SCF¹⁻¹⁸³ plasmid(Example 6B) which had been cut with HindIII, end filled with the Klenowfragment of DNA polymerase 1 (to yield a blunt end), and then cut withSpe1 and gel purified. To allow for site-directed mutagenesis as above,the SCF¹⁻²⁴⁸ fragment was cloned into MP11 from Xba1 to BamH1; analogplasmids encoding initiating methionine followed by amino acids 1-189,1-188, 1-185, or 1-180 (using numbering of FIG. 42) were then made usingsite-directed mutagenesis.

A plasmid with initiating methionine codon followed by codons for aminoacids 1 to 220 (using the numbering of FIG. 44) was made using DNAdirectly from the cDNA clone (Example 18), using the same methodsoutlined in the preceding paragraph. Similarly, analog plasmids encodinginitiating methionine followed by amino acids 1-161, 1-160, 1-157, or1-152 (using the numbering of FIG. 44) were made.

A pCFM1156 human SCF²⁻¹⁶⁵ plasmid was made by cloning the Xba1 to EcoR1SCF fragment from pCFM1156 human SCF²⁻¹⁶⁴ into the plasmid pCFM1156human SCF¹⁻¹⁶⁵ (having synthetic codons; see Example 6B). Both DNAs werecut with Xba1 and EcoR1 and the fragments gel purified for cloning. Thesmall fragment from pCFM1156 human SCF²⁻¹⁶⁴ was ligated to the largefragment of pCFM1156 human SCF¹⁻¹⁶⁵ (synthetic codons).

In considering the analog plasmids described above, it is noted thatamino acids 4, 43, 89, and 138 are Cys in human SCFs, and the codons forCys-4 or Cys-138 are missing in certain of the plasmids described. Aminoacids of the hydrophobic transmembrane region are at positions 190(about) to 212 in the numbering of FIG. 42, and positions 162 (about) to184 in the numbering of FIG. 44. Thus most of the plasmids describedencode amino acids that would be in the extracellular domain of membranebound human SCF¹⁻²⁴⁸ (FIG. 42 numbering) or human SCF¹⁻²²⁰ (FIG. 44numbering), and some include virtually all of these extracellulardomains.

Plasmids encoding various other human SCF analogs and fragments can alsobe prepared by the methods described, and by other methods known tothose skilled in the art. These include plasmids with codons for Cysresidues replaced by codons for other amino acids such as Ser.

E. coli host strain FM5 (Example 6) has been transformed with many ofthe analog plasmids described. These strains have been grown, withtemperature induction, in flasks, and in fermentors as described inExample 6C.

After fermentation and harvesting of cells, many folded, oxidized,purified SCF analogs have been recovered by the methods outlined inExample 10. These include (by the numbering of FIG. 42) SCF¹⁻¹⁸⁹,SCF¹⁻¹⁸⁸, SCF¹⁻¹⁸⁵, SCF¹⁻¹⁸⁰, SCF¹⁻¹⁵⁶, SCF¹⁻¹⁴¹, SCF¹⁻¹³⁷, SCF¹⁻¹³⁰,SCF²⁻¹⁶⁴, SCF⁵⁻¹⁶⁴, SCF¹¹⁻¹⁶⁴, and (by the numbering of FIG. 44)SCF¹⁻¹⁶¹, SCF¹⁻¹⁶⁰, SCF¹⁻¹⁵⁷, SCF¹⁻¹⁵². Like SCF¹⁻¹⁶⁴ and SCF¹⁻¹⁶⁵(Examples 17 and 27), these analogs are all dimeric in solution, asjudged using gel filtration. Most of these have biological specificactivities in the radioreceptor assay (Example 9) and UT-7 proliferationassay (Example 9) similar to those of SCF¹⁻¹⁶⁴ and SCF¹⁻¹⁶⁵ (Example 9).Some, such as SCF²⁻¹⁶⁴ and SCF⁵⁻¹⁶⁴ have lowered specific activities inthe radioreceptor assay and/or UT-7 assay (30-80% of the values forSCF¹⁻¹⁶⁴ and SCF¹⁻¹⁶⁵) while others, such as SCF¹¹⁻¹⁶⁴, have negligiblespecific activity in both assays. SCF¹⁻¹³⁰ has lowered specific activityin both the radioreceptor assay (about 50% of the value for SCF¹⁻¹⁶⁴)and the UT-7 assay (about 15% of the value for SCF¹⁻¹⁶⁴). SCF¹⁻¹³⁷ hasfull specific activity in the radioreceptor assay but lowered specificactivity in the UT-7 assay (about 25% of the value for SCF¹⁻¹⁶⁴ andSCF¹⁻¹⁶⁵); this analog therefore may be preferable as an SCF antagonistin situations where it would be advantageous to block the biologicalactivity of SCF.

While the present invention has been described in terms of preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations which come withinthe scope of the invention as claimed.

What is claimed is:
 1. An isolated DNA sequence encoding a polypeptideproduct having the hematopoietic biological activity of stimulatinggrowth of early hematopoietic progenitor cells, said DNA sequenceselected from the group consisting of: (a) DNA sequences set forth FIG.15B, FIG. 15C, FIG. 42A-D, FIG. 44A-C, or their complementary strands;(b) DNA sequences which hybridize to the DNA sequences defined in (a) orfragments thereof, or DNA sequences whose coding regions hybridize tothe DNA sequences defined in (a) or fragments thereof, the polypeptidefragment encoded by said DNA possessing hematopoietic biologicalactivity, wherein hybridization conditions are defined as (i) bringingtogether said hybridizing DNA with said DNA of subpart (a) or fragmentsthereof in the presence of 1% SDS and 6×SSC at 62° C. for an appropriatelength of time; and removing unhybridized DNA in the presence 1% SDS and6×SSC at 62° C.; or (ii) more stringent conditions; and, (c) isolatedDNA sequences differing from the isolated DNAs of (a) or (b) above innucleotide sequence due to the degeneracy of the genetic code, and whichencode a polypeptide product having the hematopoietic biologicalactivity of stimulating growth of early hematopoietic progenitor cells.2. A cDNA sequence according to claim
 1. 3. A genomic DNA sequenceaccording to claim
 1. 4. A DNA sequence according to claim 1 whereinsaid DNA sequence encodes human stem cell factor.
 5. A DNA sequenceaccording to claim 1 including one or more codons preferred forexpression in E. coli cells.
 6. A DNA sequence according to claim 1including one or more codons preferred for expression in yeast cells. 7.A DNA sequence according to claim 1 covalently associated with adetectable label substance.
 8. A DNA sequence according to claim 1 whichis single stranded.
 9. A biologically functional DNA vector including aDNA molecule according to claim
 1. 10. A procaryotic or eucaryotic hostcell stably transformed or transfected with a DNA vector according toclaim 9 in a manner allowing the host cell to express said polypeptideproduct.
 11. A process for the production of a polypeptide producthaving the hematopoietic biological activity of stimulating growth ofearly hematopoietic cells, said process comprising: growing, undersuitable nutrient conditions, procaryotic or eucaryotic host cellstransformed or transfected with a DNA molecule according to claim 1 in amanner allowing expression of said polypeptide product, and isolatingdesired polypeptide product of the expression of said DNA molecule. 12.A purified and isolated DNA sequence selected from the group consistingof DNA sequences set forth in FIG. 15B, FIG. 15C, FIG. 42A-D, and FIG.44A-C.
 13. An isolated DNA sequence encoding a polypeptide selected fromthe group consisting of, with respect to FIG. 15C, polypeptides withamino acid sequences 1-165, 1-164, and 1-162, said polypeptidesoptionally having an additional methionine at the N-terminus.
 14. Anisolated DNA sequence encoding a polypeptide selected from the groupconsisting of, with respect to FIG. 42A-C, polypeptides with amino acidsequences 1-100, 1-110, 1-120, 1-123, 1-127, 1-130, 1-133, 1-137, 1-141,1-145, 1-148, 1-152, 1-156, 1-157, 1-158, 1-159, 1-160, 1-161, 1-163,1-166, 1-168, 1-173, 1-178, 2-164, 2-165, 5-164, 11-164, 1-180, 1-183,1-185, 1-188, 1-189, 1-220, and 1-248 said polypeptides optionallyhaving an additional methionine at the N-terminus.
 15. An isolated DNAsequence encoding a polypeptide selected from the group consisting of,with respect to FIG. 44A-C, polypeptides with amino acid sequences1-220, 1-161, 1-160, 1-157 and 1-152, said polypeptides optionallyhaving an additional methionine at the N-terminus.
 16. A biologicallyfunctional DNA vector including a DNA molecule according to claims 13,14, or
 15. 17. A procaryotic or eucaryotic host cell stably transformedor transfected with a DNA vector according to claim 16 in a mannerallowing the host cell to express said polypeptide product.